Methods and compositions relating to improved lentiviral vectors and their applications

ABSTRACT

The present invention provides HIV-derived lentivectors which are safe, highly efficient, and very potent for expressing transgenes for human gene therapy, especially, in human hematopoietic progenitor cells as well as in all other blood cell derivatives. The lentiviral vectors comprise a self-inactivating configuration for biosafety and promoters such as the EF1α promoter as one example. Additional promoters are also described. The vectors can also comprise additional transcription enhancing elements such as the wood chuck hepatitis virus post-transcriptional regulatory element. These vectors therefore provide useful tools for genetic treatments such as inherited and acquired lympho-hematological disorders, gene-therapies for cancers especially the hematological cancers, as well as for the study of hematopoiesis via lentivector-mediated modification of human HSCs.

The present application is a continuation of U.S. patent applicationSer. No. 15/263,027, filed Sep. 12, 2016, now U.S. Pat. No. 9,731,033,which is a continuation of U.S. patent application Ser. No. 14/693,406,filed Apr. 22, 2015, now U.S. Pat. No. 9,476,062, which is acontinuation of U.S. patent application Ser. No. 14/022,121, filed Sep.9, 2013, now U.S. Pat. No. 9,023,646, which is a continuation of U.S.patent application Ser. No. 13/622,309, filed Sep. 18, 2012, now U.S.Pat. No. 8,551,773, which is a continuation of U.S. patent applicationSer. No. 12/537,789, filed Aug. 7, 2009, now U.S. Pat. No. 8,329,462,which is a continuation of U.S. patent application Ser. No. 10/010,081,filed Nov. 9, 2001, now U.S. Pat. No. 7,575,924, which claims thebenefit of U.S. Provisional Application Ser. No. 60/248,398 filed onNov. 13, 2000. The entire contents of each of the above-referencedapplications are hereby incorporated by reference.

BACKGROUND OF THE INVENTION 1. Field of the Invention

The present invention relates to improved lentiviral vectors and theiruse in gene delivery and high level expression of desired transgenes totarget cells, particularly to human hematopoietic progenitor cells anddifferentiated blood lineages.

2. Description of Related Art

Gene therapy via the transduction of human hematopoietic stem cells(hHSC) represents a very promising approach for the treatment of anumber of inherited and acquired lympho-hematological disorders. Thestable genetic manipulation of long term repopulating hHSC with existinggene delivery systems, however, has been impossible to achieve at anefficiency compatible with therapeutic realities. Oncoretroviral vectorsderived from Moloney murine leukemia virus (MLV), for instance, althoughhighly appealing since they integrate their cargo into the chromosomesof target cells, cannot transduce hHSC that have not been first treatedwith inducers of proliferation (Kohn et al., 1991; Mazurier et al.,1998). Indeed, the nuclear transport of the MLV preintegration complexrequires the breakdown of the nuclear envelope that occurs at mitosis(Roe et al., 1993; Lewis and Emerman, 1994). Unfortunately hHSCs,whether harvested from the bone marrow (BM), the umbilical cord (UC) ormobilized in the peripheral circulation, are mostly quiescent and losetheir pluripotentiality after stimulation and proliferation (Bhatia etal., 1997; Dao et al., 1997; Dorrell et al., 2000). Recent reports,however, have shown that a significant fraction of pluripotent cells aswell as cells capable of long-term engraftment in non-obesediabetic/severe combined immunodeficient (NOD/SCID), also calledSCID-repopulating cells (SRC), can be maintained, transduced and evenexpanded using specific stimulation conditions (Dorrell et al., 2000;Dao et al., 1998; Piacibello et al., 1999; Ueda et al., 2000).

Lentiviruses are a subgroup of retroviruses that can infect nondividingcells owing to the karyophilic properties of their preintegrationcomplex, which allow for its active import through the nucleopore.Correspondingly, lentiviral vectors derived from human immunodeficiencyvirus type 1 (HIV-1) can mediate the efficient delivery, integration andlong-term expression of transgenes into non-mitotic cells both in vitroand in vivo (Naldini et al., 1996a; Naldini et al., 1996b; Blomer etal., 1997). In particular, HIV-based vectors can efficiently transducehuman CD34⁺ hematopoietic cells in the absence of cytokine stimulation(Akkina et al., 1996; Sutton et al., 1998; Uchida et al., 1998; Miyoshiet al., 1999; Case et al., 1999), and these cells are capable oflong-term engraftment in NOD/SCID mice (Miyoshi et al., 1999).Furthermore, bone marrow from these primary recipients can repopulatesecondary mice with transduced cells, confirming thelentivector-mediated genetic modification of very primitivehematopoietic precursors, most probably bona fide stem cells. Since noneof the other currently available gene delivery systems has such anability, lentiviral vectors provide a previously unexplored basis forthe study of hematopoiesis and for the gene therapy of inherited andacquired lympho-hematopoietic disorders via the genetic modification ofHSCs.

The demonstration of this important point, however, was provided with anearly generation of lentiviral vectors unsuitable for therapeuticapplications, either because they failed to meet biosafety requirements(Akkina et al., 1996; Sutton et al., 1998; Uchida et al., 1998) orbecause they induced levels of transgene expression that weredismissingly low (Miyoshi et al., 1999; Case et al., 1999; An et al.,2000). Accordingly, there is a significant need to develop improvedlentiviruses for use as transducing vectors that are capable ofeffectively transducing hematopoietic cells, particularly hematopoieticprogenitor cells, and which are capable of expressing desired transgenesat high levels.

SUMMARY OF THE INVENTION

The present invention is directed to the development of improvedlentiviral vectors that both meet biosafety requirements, and whichinduce high levels of transgene expression. Accordingly, the presentinvention describes gene transfer vehicles that appear particularly wellsuited for the transduction of human hematopoietic precursor cells(HPCs) and for the expression of transgenes in differentiated bloodlineages. These vectors will facilitate the further use of lentiviralvectors for the genetic manipulation of lympho-hematopoietic cells, andshould be particularly useful for both research and therapeuticapplications. However, it will be understood by the skilled artisan thatthe invention is not limited to the transduction of hematopoietic cellsand that one may use the lentiviral vectors of the invention for theexpression of transgenes in other cell types as well. Some examples ofother cell types contemplated include terminally differentiated cellssuch as neurons, lung cells, muscle cells, liver cells, pancreaticcells, endothelial cells, cardiac cells, skin cells, bone marrow stromalcells, and eye cells. Additionally, stem cells and progenitor cells suchas pancreatic ductal cells, neural precursors, and mesodermal stem cellsare also contemplated.

The present invention thus concerns, in a general and overall sense,improved vectors that are designed to permit the transfection andtransduction of human hematopoietic progenitor cells, or stem cells(hHSC), and provide high level expression of desired transgenes in suchcells. The vectors of the present invention may be referred to asself-inactivating lentivectors due to the presence of lentiviralelements as well as certain “self-inactivating” design characteristicsthat render these vectors safe for human applications.

The lentivectors of the present invention provide, for the first time,an efficient means of achieving high level expression of desiredtrangenes in hHSCs, cells which have been difficult to transfect andtransduce due to the fact that in their unstimulated state, they arerelatively resistant to transduction by previous vector systems. Theselentivectors have the ability to infect non-dividing cells owing to thekaryophilic properties of their preintegration complex, which allow forits active import through the nucleopore. Moreover, preferred lentiviralvectors of the present invention can mediate the efficient delivery,integration and long-term expression of transgenes into non-mitoticcells both in vitro and in vivo, even in the absence of cytokinestimulation. Stem cells transduced by the more preferred lentivectors ofthe present invention are capable of long-term engraftment, for example,in NOD/SCID mice. Most notably, however, the more preferred lentivectorsof the present invention have highly desirable features that permit thehigh level expression of transgenes in human progenitor cells whilemeeting human biosafety requirements.

The viral vectors of the present invention, therefore, may be generallydescribed as self-inactivating recombinant vectors that include at leasta lentiviral gag, pol and rev genes, that is, those genes required forvirus production, which permit their manufacture in reasonablequantities using available producer cell lines. To meet important humansafety needs, the more preferred vectors in accordance with the presentinvention will not include any other active lentiviral genes, such asvpr, vif, vpu, nef, tat, such as where these genes have been removed orotherwise inactivated. In fact, it is preferred that the only activelentiviral genes present in the vector will be the aforementioned gag,pol and rev genes.

The most preferred lentiviral genes and backbone (i.e., long terminalrepeats or LTRs) used in preparing lentivectors in accordance with thepresent invention will be one that is human immunodeficiency virus (HIV)derived, and more particularly, HIV-1 derived. Thus, the gag, pol andrev genes will preferably be HIV genes and more preferably HIV-1 genes.However, the gag, pol and rev genes and LTR regions from otherlentiviruses may be employed for certain applications in accordance withthe present invention, including the genes and LTRs of HIV-2, simianimmunodeficiency virus (SIV), feline immunodeficiency virus, bovineimmunodeficiency virus, equine infectious anemia virus, caprinearthritis encephalitis virus and the like. Such constructs could beuseful, for example, where one desires to modify certain cells ofnon-human origin. However, the HIV based vector backbones (i.e., HIV LTRand HIV gag, pol and rev genes) will generally be preferred inconnection with most aspects of the present invention in that HIV-basedconstructs are the most efficient at transduction of human hematopoieticprogenitor cells.

The viral vectors of the present invention will also include anexpression cassette comprising a transgene positioned under the controlof a promoter that is active to promote detectable transcription of thetransgene in a human hematopoietic progenitor cell. To determine whethera particular promoter is useful, a selected promoter is tested in theconstruct in vitro in a selected progenitor cell and, if the promoter iscapable of promoting expression of the transgene at a detectablesignal-to-noise ratio, it will generally be useful in accordance withthe present invention. A desirable signal-to-noise ratio is one betweenabout 10 and about 200, a more desirable signal-to-noise ratio is one 40and about 200, and an even more desirable signal-to-noise ratio is onebetween about 150 and about 200. One means of testing such a promoter,described in more detail hereinbelow, is through the use of a signalgenerating transgene such as the green fluorescent protein (GFP).

Examples of promoters that may be preferably employed in connection withthe present invention including an EF1-α, PGK, gp91hox, MHC classII,clotting Factor IX, insulin promoter, PDX1 promoter, CD11, CD4, CD2 or agp47 promoter. Of these the PGK promoter is preferred, and the EF1-αpromoter is particularly preferred. An example of a promoter that isgenerally not preferred is the CMV promoter, in that this promoter isonly minimally active in most progenitor cells. Indeed, it is likelythat a CMV promoter will only be useful where one targets human primaryB cells or dendritic cells. In any event, however, practice of thepresent invention is not restricted to the foregoing promoters, so longas the promoter is active in the progenitor, hematopoietic or other cellthat one desires to target.

Additionally, the promoters mentioned above can comprise additionalelements required for transcription and thus be a part of atranscription cassette. A transcription cassette is defined ascomprising one or more promoter elements coupled to enhancers and/orlocus control regions, to ensure strong and/or tissue-restrictedexpression of a transgene.

It is particularly desirable to employ in the lentivectors of thepresent invention an LTR region that has reduced promoter activityrelative to wild-type LTR, in that such constructs provide a“self-inactivating” (SIN) biosafety feature. Self-inactivating vectorsare ones in which the production of full-length vector RNA in transducedcells in greatly reduced or abolished altogether. This feature greatlyminimizes the risk that replication-competent recombinants (RCRs) willemerge. Furthermore, it reduces the risk that that cellular codingsequences located adjacent to the vector integration site will beaberrantly expressed. Furthermore, an SIN design reduces the possibilityof interference between the LTR and the promoter that is driving theexpression of the transgene. It is therefore particularly suitable toreveal the full potential of the internal promoter.

Self-inactivation is preferably achieved through in the introduction ofa deletion in the U3 region of the 3′ LTR of the vector DNA, i.e., theDNA used to produce the vector RNA. Thus, during reverse transcription,this deletion is transferred to the 5′ LTR of the proviral DNA. It isdesirable to eliminate enough of the U3 sequence to greatly diminish orabolish altogether the transcriptional activity of the LTR, therebygreatly diminishing or abolishing the production of full-length vectorRNA in transduced cells. However, it is generally desirable to retainthose elements of the LTR that are involved in polyadenylation of theviral RNA, a function spread out over U3, R and U5. Accordingly, it isdesirable to eliminate as many of the transcriptionally important motifsfrom the LTR as possible while sparing the polyadenylation determinants.In the case of HIV based lentivectors, it has been discovered that suchvectors tolerate significant U3 deletions, including the removal of theLTR TATA box (e.g., deletions from −418 to −18), without significantreductions in vector titers. These deletions render the LTR regionsubstantially transcriptionally inactive in that the transcriptionalability of the LTR in reduced to about 90% or lower. In preferredembodiments the LTR transcription is reduced to about 95% to 99%. Thus,the LTR may be rendered about 90%, 91%, 92%, 93%, 94%, 95% 96% 97%, 98%,to about 99% transcriptionally inactive.

For certain applications, for example, in the case of promoters that areonly modestly active in cells targeted for transduction, one will desireto employ a posttranscriptional regulatory sequence positioned topromote the expression of the transgene. One type of posttranscriptionalregulatory sequence is an intron positioned within the expressioncassette, which may serve to stimulate gene expression. However, intronsplaced in such a manner may expose the lentiviral RNA transcript to thenormal cellular splicing and processing mechanisms. Thus, in particularembodiments it may be desirable to locate intron-containing transgenesin an orientation opposite to that of the vector genomic transcript.

A more preferred method of enhancing transgene expression is through theuse of a posttranscriptional regulatory element which does not rely onsplicing events, such as the posttranscriptional processing element ofherpes simplex virus, the posttranscriptional regulatory element of thehepatitis B virus (HPRE) or that of the woodchuck hepatitis virus(WPRE), which contains an additional cis-acting element not found in theHPRE. The regulatory element is positioned within the vector so as to beincluded in the RNA transcript of the transgene, but outside of stopcodon of the transgene translational unit. It has been found that theuse of such regulatory elements are particularly preferred in thecontext of modest promoters, but may be contraindicated in the case ofvery highly efficient promoters.

It is believed that the lentivectors of the present invention may beemployed to deliver any transgene that one desires, depending on theapplication. In the case of delivery to hematopoietic progenitor cells,one will typically select a transgene that will confer a desirablefunction on such cells, including, for example, globin genes,hematopoietic growth factors, which include erythropoietin (EPO), theinterleukins (such as Interleukin-1 (IL-1), Interleukin-2 (IL-2),Interleukin-3 (IL-3), Interleukin-6 (IL-6), Interleukin-12 (IL-12),etc.) and the colony-stimulating factors (such as granulocytecolony-stimulating factor, granulocyte/macrophage colony-stimulatingfactor, or stem-cell colony-stimulating factor), the platelet-specificintegrin αIIbβ, multidrug resistance genes, the gp91 or gp 47 genes thatare defective in patients with chronic granulomatous disease (CGD),antiviral genes rendering cells resistant to infections with pathogenssuch as human immunodeficiency virus, genes coding for blood coagulationfactors VIII or IX which are mutated in hemophiliacs, ligands involvedin T cell-mediated immune responses such as T cell antigen receptors, Bcell antigen receptors (immunoglobulins) as well as combination of T andB cell antigen receptors alone or in combination with single chainantibodies such as ScFv, tumor necrosis factor (TNF), IL-2, IL-12, gammainterferon, CTLA4, B7 and the like, genes expressed in tumor cells suchas Melana, MAGE genes (such as MAGE-1, MAGE-3), P198, P1A, gp100 etc.

A principal application of the present transgenes will be to deliverdesired transgenes to hematopoietic cells for a number of possiblereasons. This might include, but of course not be limited to, thetreatment of myelosupression and neutropenias which may be caused as aresult of chemotherapy or immunosupressive therapy or infections such asAIDS, genetic disorders, cancers and the like.

Exemplary genetic disorders of hematopoietic cells that are contemplatedinclude sickle cell anemia, thalassemias, hemaglobinopathies, Glanzmannthrombasthenia, lysosomal storage disorders (such as Fabry disease,Gaucher disease, Niemann-Pick disease, and Wiskott-Aldrich syndrome),severe combined immunodeficiency syndromes (SCID), as well as diseasesresulting from the lack of systemic production of a secreted protein,for example, coagulation factor VIII and/or IX. In such cases, one woulddesire to introduce transgenes such as globin genes, hematopoieticgrowth factors, which include erythropoietin (EPO), the interleukins(especially Interleukin-1, Interleukin-2, Interleukin-3, Interleukin-6,Interleukin-12, etc.) and the colony-stimulating factors (such asgranulocyte colony-stimulating factor, granulocyte/macrophagecolony-stimulating factor, or stem-cell colony-stimulating factor), theplatelet-specific integrin αIIbβ, multidrug resistance genes, the gp91or gp 47 genes which are defective in patients with chronicgranulomatous disease (CGD), antiviral genes rendering cells resistantto infections with pathogens such as human immunodeficiency virus, genescoding for blood coagulation factors VIII or IX which are mutated inhemophiliacs, ligands involved in T cell-mediated immune responses suchas T cell antigen receptors, B cell antigen receptors (immunoglobulins),a combination of both T and B cell antigen receptors alone and/or incombination with single chain antibodies (ScFv), IL2, IL12, TNF, gammainterferon, CTLA4, B7 and the like, genes expressed in tumor cells suchas Melana, MAGE genes (such as MAGE-1, MAGE-3), P198, P1A, gp100 etc.

Exemplary cancers are those of hematopoietic origin, for example,arising from myeloid, lymphoid or erythroid lineages, or precursor cellsthereof. Exemplary myeloid disorders include, but are not limited to,acute promyeloid leukemia (APML), acute myelogenous leukemia (AML) andchronic myelogenous leukemia (CML). Lymphoid malignancies which may betreated utilizing the lentivectors of the present invention include, butare not limited to acute lymphoblastic leukemia (ALL) which includesB-lineage ALL and T-lineage ALL, chronic lymphocytic leukemia (CLL),prolymphocytic leukemia (PLL), hairy cell leukemia (HLL) andWaldenstrom's macroglobulinemia (WM). Additional forms of malignantlymphomas contemplated as candidates for treatment utilizing thelentiviral vectors of the present invention include, but are not limitedto non-Hodgkin lymphoma and variants thereof, peripheral T-celllymphomas, adult T-cell leukemia/lymphoma (ATL), cutaneous T-celllymphoma (CTCL), large granular lymphocytic leukemia (LGF) and Hodgkin'sdisease.

In other embodiments, the present invention is directed to host cellsthat have been transduced with one of the foregoing lentivectors. It isbelieved that the lentivectors of the present invention can be employedto transduce most any cell. Exemplary cells include but are not limitedto a CD4⁺ T cell, a peripheral blood lymphocyte cell, a peripheral bloodmononuclear cell, a hematopoietic stem cell, a fetal cord blood cell, afibroblast cell, a brain cell, a lung cell, a liver cell, a muscle cell,a pancreatic cell, an endothelial cell, a cardiac cell, a skin cell, abone marrow stromal cell, and an eye cells, a pancreatic ductal cell, aneural precursor, a mesodermal stem cell and the like. The cellstransduced may further be primate, murine, porcine, or human in origin,or come from another animal species.

For the production of virus particles, one may employ any cell that iscompatible with the expression of lentiviral Gag and Pol genes, or anycell that can be engineered to support such expression. For example,producer cells such as 293T cells and HT1080 cells may be used.

Of course, as noted above, the lentivectors of the invention will beparticularly useful in the transduction of human hematopoieticprogenitor cell or a hematopoietic stem cell, obtained either from thebone marrow, the peripheral blood or the umbilical cord blood, as wellas in the transduction of a CD4⁺ T cell, a peripheral blood B or Tlymphocyte cell, a peripheral blood mononuclear cell, a dendritic cell,and a monocytic cell. Particularly preferred targets are CD34⁺ cells.

In still other embodiments, the present invention is directed to amethod for transducing a human hematopoietic stem cell comprisingcontacting a population of human cells that include hematopoietic stemcells with one of the foregoing lentivectors under conditions to effectthe transduction of a human hematopoietic progenitor cell in saidpopulation by the vector. The stem cells may be transduced in vivo or invitro, depending on the ultimate application. Even in the context ofhuman gene therapy, such as gene therapy of human stem cells, one maytransduce the stem cell in vivo or, alternatively, transduce in vitrofollowed by infusion of the transduced stem cell into a human subject.In one aspect of this embodiment, the human stem cell can be removedfrom a human, e.g., a human patient, using methods well known to thoseof skill in the art and transduced as noted above. The transduced stemcells are then reintroduced into the same or a different human.

Where a human subject is treated directly by introduction of the vectorinto the subject, the treatment is typically carried out by intravenousadministration of the vector. When cells, for instance CD34⁺ cells,dendritic cells, peripheral blood cells or tumor cells are transduced exvivo, the vector particles are incubated with the cells using a dosegenerally in the order of between 1 to 50 multiplicities of infection(MOI) which also corresponds to 1×10⁵ to 50×10⁵ transducing units of theviral vector per 10⁵ cells. This of course includes amount of vectorcorresponding to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40,45, and 50 MOI. Typically, the amount of vector may be expressed interms of HeLa transducing units (TU). Other routes for vectoradministration include intrarterially, endoscopically, intralesionally,percutaneously, subcutaneously, intramuscular, intrathecally,intraorbitally, intradermally, intraperitoneally, transtracheally,subcuticularly, by intrasternal injection, by inhalation or intranasalspraying, by endrotracheal route and the like. In embodiments concerningtumor/cancer therapies with the vectors of the invention the expressionvector can be delivered by direct injection into the tumor or into thetumor vasculature.

A typical example of ex vivo gene therapy is a patient suffering fromchronic granulatous disease (CGD), whose CD34⁺ cells can be isolatedfrom the bone marrow or the peripheral blood and transduced ex vivo witha lentivector expressing the gp91hox gene before reimplantation. In thecase of patients suffering from severe combined immunodeficiency (SCID),the inventors contemplate a similar approach, using lentivectors of theinvention expressing the gene defective in the patient, for example, thegene encoding the common gamma chain of the Interleukin receptor. Forthe genetic treatment of HIV infection, the present inventorscontemplate intracellular immunization, wherein cells are renderedresistant to the HIV virus through the introduction of antiviral genes.In embodiments of the intracellular immunization for HIV, targets of thelentivectors of the invention include hematopoietic progenitors,peripheral blood CD4⁺ T cells, and monocytes. As will be recognized bythe skilled artisan, similar intracellular immunization methods can beused for other viral infections as well. For the immunotherapy ofcancers, tumor cells or antigen presenting cells such as dendritic cellswill be genetically engineered with the lentivectors of the invention.For cancer therapies some transgenes that may be used in the lentivectorconstructs of the invention are those that can inhibit, and/or kill,and/or prevent the proliferation, and/or mediate the apoptosis of, thecancer/tumor cell and/or genes such as TNF.

The lentivectors described herein may also be used in vivo, by directinjection into the blood or into a specific organ. For example, in oneembodiment intracerebral injection of lentivectors expressing the GlialCell Derived Nerve Growth Factor (GDNF), can be used for the treatmentof Parkinson's disease. In another example, intraportal injection of alentivector expressing coagulation factor VIII for the correction ofhemophilia A is envisioned. In yet another example, intravenous orintramuscular injection of a lentivector of the present inventionexpressing the dystrophin gene for the treatment of Duchenne MuscularDystrophy is envisioned. Thus, one of ordinary skill in the art willappreciate the extensive use of the lentivector constructs of thepresent invention in terms of gene therapies.

As used herein the specification or claim(s) when used in conjunctionwith the word “comprising”, the words “a” or “an” may mean one or morethan one. As used herein “another” may mean at least a second or more.

Other objects, features and advantages of the present invention willbecome apparent from the following detailed description. It should beunderstood, however, that the detailed description and the specificexamples, while indicating preferred embodiments of the invention, aregiven by way of illustration only, since various changes andmodifications within the spirit and scope of the invention will becomeapparent to those skilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings form part of the present specification and areincluded to further demonstrate certain aspects of the presentinvention. The invention may be better understood by reference to one ormore of these drawings in combination with the detailed description ofspecific embodiments presented herein.

FIG. 1A and FIG. 1B. Transgene transfer and expression aftertransduction of human CD34⁺ cells with MLV and HIV vectors. Human CD34⁺cells from umbilical cord blood (10⁵ cells) were transduced with 10⁶HeLa-transducing units (TU) of MLV- or the indicated HIV-derived GFPencoding vectors, previously treated with DNAse I, as described inmaterials and methods. After 4 days, cells were analyzed by flowcytometry for GFP expression (FIG. 1A) and lysed for PCR analysis of thepresence of GFP transgene (FIG. 1B). Results from one experimentrepresentative of two independent evaluations are shown. Expression ofthe GFP transgene after transduction of human CD34⁺ cells or HeLa cells.Results are represented as histograms of GFP fluorescence intensity(x-axis, four-log scale) versus cell number (y-axis, linear). GFP⁺ cells(within marker) were analyzed for percentage (lower number) and medianof fluorescence intensity (upper number). Percentages were omitted inHeLa cells since the histograms were obtained from titration experimentsof the corresponding vectors (FIG. 1A). Presence of the GFP transgeneafter transduction and expansion of human CD34⁺ cells. Cellular extractsequivalent to 5000 cells (upper panel) were amplified as described inthe section entitled Examples with GFP-specific primers, together withIL-2 specific primers as internal controls. Sizes of the correspondingPCR products are indicated. Lanes are: M, molecular weight marker; HeLa(negative control for GFP, positive control for IL-2); 4.5, a clone ofHeLa containing one copy of HIV-CMV-GFP vector; 0, untransduced CD34⁺cells; MLV-CMV-PGK-EF1, CD34⁺ cells transduced with the correspondingvectors (see FIG. 1A); CMV-EPO, CD34⁺ cells transduced with HIV-CMVvector (lane CMV) and analyzed after expansion and differentiation intoerythroid cells (see text); CMV-GM, same as CMV-EPO but analyzed afterexpansion and differentiation into monocytic cells (see text); PGK-EPOand PGK-GM, same as CMV-EPO and CMV-GM but from CD34+ cells transducedwith HIV-CMV vector (lane PGK). To ensure for proportionality, cellularextracts equivalent to 1700 cells (lower panel) were amplifiedseparately (FIG. 1B).

FIG. 2. Effect of MOI on transduction efficiency of lentivectors inhuman CD34⁺ cells. 10⁵ CD34⁺ cells were transduced with various doses(1, 2, 5, 10, 20 and 50×10⁵ TU) of EF1α-GFP lentiviral vector allowingmultiplicities of infection (MOI) ranging from 1 to 50. GFP expressionwas analyzed by flow cytometry after 4 days. Upper panel: Data from onerepresentative experiment are shown as frequency histograms of GFPfluorescence intensity versus cell number (events). Gates for GFP⁺ cellswere setup according to untransfected cells (MOI 0). Lower panel: Meanpercentages of GFP⁺ cells obtained with MOIs of 0 to 10 and 10 to 50 arerepresented in the main panel and in the insert respectively. These dataare from 3 independent experiments for the 0-10 MOI range and from 2 for10-50 MOI range. Error bars represent SD.

FIG. 3. Transgene expression in differentiated hematopoietic lineagesafter transduction of human CD34⁺ cells with HIV-derived vectors. HumanCD34⁺ cells from umbilical cord blood (10⁵ cells) were transduced with10⁶ TU of HIV vectors (corresponding to a MOI of 10) containing eitherthe EF1α or the PGK promoters and differentiated into varioushematopoietic lineages as described in the section entitled Examples.After differentiation, HIV-EF1α- (first and second column) orHIV-PGK-transduced cells (third column) were analyzed by flow cytometryfor both GFP (x axis) and lineage-specific markers expression(glycophorin, CD14, CD42b, CD15 and CD1a for respectively erythroids,monocytes, megakaryocytes, granulocytes, and dendritic cells). Isotypecontrol antibodies were used in the first column. For each promoter,cell populations expressing high levels of lineage-specific marker weregated (upper rectangle in 2D plots), and monoparametric frequencyhistograms of GFP expression for these cells were generated (locatedabove the 2D plots). The percentage of GFP⁺ cells (upper number), andmedian of fluorescence intensity of GFP (lower number) was determined.For the experiment generating erythroids, monocytes, megakaryocytes andgranulocytes, the fraction of GFP⁺ CD34⁺ cells before differentiationwas 22% for EF1α and 16% for PGK. For the experiment generating DCs, thefraction of GFP⁺ precursors before differentiation was 32% for EF1α and29% for PGK. These data are representative of 4 independent experiments.

FIG. 4. GFP expression in lentivector-transduced primary human Tlymphocytes. CMV-, PGK- or EF1α-containing HIV-derived vectors were usedto infect PHA-activated primary T lymphocytes. Cells were maintained for5 days in culture medium supplemented with IL-2 before analyzing GFPexpression by flow cytometry. Two-dimensional plot represents cellnumber as a function of GFP levels. Results are representative of atleast five independent experiments.

FIG. 5A. and FIG. 5B. Effect of SIN design and WPRE addition ontransgene expression in human CD34⁺ cells. 10⁵ CD34⁺ cells weretransduced with 10⁶ HeLa-TU of the indicated GFP-expressing HIV-derivedvectors and analyzed by flow cytometry four days later. Vectors were asfollows: (FIG. 5A) PGK (2 intact LTRs); PGK-SIN (3′ SIN LTR deletion,see materials and methods); PGK-SIN-W (addition of WPRE upstream of the3′SIN-LTR). (FIG. 5B) Same analysis as in FIG. 5A, but with EF1αpromoter instead of PGK promoter. Results are represented as contourgraphs of CD34 expression versus GFP expression (four-log scale). Foreach condition, GFP expression is also displayed as histograms of GFPfluorescence intensity (x-axis, four-log scale) versus number of cells(y-axis, linear). Number in square represents median of fluorescenceintensity of GFP⁺ cells.

FIG. 6. Map depicting the construct pHR-CMV-GFP. Same as pHR-CMV-LACz(Genbank AF 105229) but with EGFP instead of LacZ.

FIG. 7. Map depicting the construct pHR-EF1-GFP. Also referred to aspHR′-EF1alpha-EGFP. ClaI, HindIII and EcoRI cut outside of EF1alphapromoter (Nagata, G181962).

FIG. 8. Map depicting the construct pHR-EF1-GFP-SIN. Deletion of WPRE byinsertion of Asp718/Asp718 fragment (˜0.8 kb) from pHR′-EFI alpha-EGFP(Uni-Lund Lab, Sweden) into Asp718/Asp718 sites of pHR-E-GFP-W.

FIG. 9. Map depicting the construct pHR-EF1-GFP-W-SIN. Insertion ofClaI/BamHI fragment from pHR′-EF1-GFP (Uni-Lund, Sweden) containing thepromoter of the human EF1 alpha gene (Nagata, G181962) into ClaI/BamHIsites of pHRGFP/delta ClaI. SalI site at 1757 destroyed for subsequentclonings.

DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

While lentiviral vectors offer a great potential for gene-therapy andespecially the transduction of human hematopoietic stem cells (hHSC),vectors developed so far have failed to meet biosaftey standards and arestill inefficient in expression of transgenes. For example, while CMVpromoter-containing HIV-derived vectors can induce high levels oftransgene expression in the central nervous system (Naldini et al.,1996a; Naldini et al., 1996b; Blomer et al., 1997), and allowed theinitial demonstration that pluripotent hematopoietic precursors can beefficiently transduced by this gene delivery tool, they are largelyuseless for transferring therapeutic genes into mostlympho-hematopoietic cells, because in these targets theirtranscriptional activity is prohibitively low (Miyoshi et al., 1999;Case et al., 1999; An et al., 2000). Current lentiviral vectors havemultiply attenuated HIV virulence genes which removes the potential forreconstitution of wild-type virus by recombination (Zufferey et al.,1997; Dull et al., 1998). A self-inactivating design rendered thevectors further biologically safe by eliminating the transcriptionalelements of HIV (Zufferey et al., 1998). However, this can negativelyaffect transgene expression, apparently by decreasing the efficiency ofpolyadenylation (DeZazzo et al., 1991; Valsamakis et al., 1991; Brown etal., 1991; Cherrington and Ganem, 1992; Valsamakis et al., 1992;Gilmartin et al., 1992).

The present invention overcomes such and other deficiencies in the artand describes the development of improved HIV-derived vectors that areoptimized in terms of both biosaftey and increased gene expression.Thus, the transduction of human cells with HIV-derived lentivectorswhich comprise elements that prevent the formation of replicationcompetent recombinants (RCR) and further comprise an internal promoterelement which induces high levels of transgene expression in bothhematopoietic precursors and in vitro differentiated blood lineages, aswell as in primary T cells demonstrated herein that. For example, humanCD34⁺ cells as well as other human hematopoietic lineages can betransduced using the vectors of this invention.

The promoter elements of the vectors described include the EF1α promoteror the PGK promoter, although, as will be recognized by one of skill inthe art, almost any promoter element may be used. However, CMV promotersare not preferred. For example, the inventors demonstrate using thegreen fluorescent protein (GFP) expression that while the EF1α, promoterelement or the PGK promoter were highly active in CD34⁺ cells as well asseveral other differentiated hematopoietic derivatives, the CMV promoterdriven GFP production was insufficient to determine either thepercentage of transduced cells or the level of transgene expression inthose cells. Also contemplated are engraftment and repopulation assaysin NOD/SCID mice with both HIV-PGK and HIV-EF1α vectors, to confirm thestability of expression from these promoters in vivo.

The element that prevents RCR in the lentivectors of the presentinvention is the self inactivating (SIN) design. This is achieved by thedeletion of a major part of U3 in the 3′LTR of the vector plasmid,leading to a self-inactivating (SIN) configuration (Zufferey et al.,1998). This deletion also prevents potential interference between LTRand the internal promoter elements. However, the SIN can inducedecreases in transgene expression, especially in promoters that are notvery strong such as the PGK promoter. The invention further describesmethods that rescue transgene levels in lentivector constructs that donot have strong promoters by inserting other regulatory elements such asthe woodchuck hepatitis virus post-transcriptional regulatory element(WPRE) or the hepatitis virus B regulatory element (HPRE) in the vector,immediately upstream of the deleted 3′ LTR.

The lentivectors of this invention can efficiently transduce severalhuman blood lineage cells including CD34⁺ cells using conditions underwhich MLV-based vectors are inefficient. Furthermore, it is alsodemonstrated that human CD34⁺ cells can be efficiently transduced at arelatively low MOI, although the efficacy of gene transfer saturates atabout 20 to 30% of transduced cells. For example, an MOI of 10 was usedto achieve optimal transduction which is significantly lower than thatdescribed in previous studies, where it ranged between 60-300 and1000-3000 (Miyoshi et al., 1999; Case et al., 1999). This may in part bedue to enhanced vector-target meeting probability as the methods of thepresent invention involve exposure of CD34⁺ cells to the vectorparticles in a small volume (10⁵ cells in 200 μl) and for a duration of24 hrs.

Thus, the present invention provides HIV-derived vectors which are safe,highly efficient, and very potent for expressing transgenes in humanhematopoietic progenitor cells as well as in all other blood cellderivatives, even in a self-inactivating configuration. These vectorstherefore provide useful tools for genetic treatments such as inheritedand acquired lympho-hematological disorders, gene-therapies for cancersespecially the hematological cancers, as well as for the study ofhematopoiesis via lentivector-mediated modification of human HSCs.

A. Lentiviral Vectors and Gene Therapy

Lentiviruses are complex retroviruses, which, in addition to the commonretroviral genes gag, pol, and env, contain other genes with regulatoryor structural function. The higher complexity enables the virus tomodulate its life cycle, as in the course of latent infection. Someexamples of lentivirus include the Human Immunodeficiency Viruses:HIV-1, HIV-2 and the Simian Immunodeficiency Virus: SIV. Lentiviralvectors have been generated by multiply attenuating the HIV virulencegenes, for example, the genes env, vif, vpr, vpu and nef are deletedmaking the vector biologically safe.

Lentiviral vectors offer great advantages for gene therapy. Theyintegrate stably into chromosomes of target cells which is required forlong-term expression. Further, they do not transfer viral genestherefore avoiding the problem of generating transduced cells that canbe destroyed by cytotoxic T-cells. Furthermore, they have a relativelylarge cloning capacity, sufficient for most envisioned clinicalapplications. In addition, lentiviruses, in contrast to otherretroviruses, are capable of transducing non-dividing cells. This isvery important in the context of gene-therapy for tissues such as thehematopoietic system, the brain, liver, lungs and muscle. For example,vectors derived from HIV-1 allow efficient in vivo and ex vivo delivery,integration and stable expression of transgenes into cells such aneurons, hepatocytes, and myocytes (Blomer et al., 1997; Kafri et al.,1997; Naldini et al., 1996; Naldini et al., 1998).

The lentiviral genome and the proviral DNA have the three genes found inretroviruses: gag, pol and env, which are flanked by two long terminalrepeat (LTR) sequences. The gag gene encodes the internal structural(matrix, capsid and nucleocapsid) proteins; the pol gene encodes theRNA-directed DNA polymerase (reverse transcriptase), a protease and anintegrase; and the env gene encodes viral envelope glycoproteins. The 5′and 3′ LTR's serve to promote transcription and polyadenylation of thevirion RNA's. The LTR contains all other cis-acting sequences necessaryfor viral replication. Lentiviruses have additional genes including vif,vpr, tat, rev, vpu, nef and vpx.

Adjacent to the 5′ LTR are sequences necessary for reverse transcriptionof the genome (the tRNA primer binding site) and for efficientencapsidation of viral RNA into particles (the Psi site). If thesequences necessary for encapsidation (or packaging of retroviral RNAinto infectious virions) are missing from the viral genome, the cisdefect prevents encapsidation of genomic RNA. However, the resultingmutant remains capable of directing the synthesis of all virionproteins.

Lentiviral vectors are known in the art, see Naldini et al., (1996 and1998); Zufferey et al., (1997); Dull et al., 1998, U.S. Pat. Nos.6,013,516; and 5,994,136 all incorporated herein by reference. Ingeneral, these vectors are plasmid-based or virus-based, and areconfigured to carry the essential sequences for incorporating foreignnucleic acid, for selection and for transfer of the nucleic acid into ahost cell.

Two components are involved in making a virus-based gene deliverysystem: first, the packaging elements, encompassing the structuralproteins as well as the enzymes necessary to generate an infectiousparticle, and second, the vector itself, i.e., the genetic material tobe transferred. Biosaftey safeguards can be introduced in the design ofboth of these components. Thus, the packaging unit of the firstgeneration HIV-based vectors comprised all HIV-1 proteins except theenvelope proteins (Naldini et al., 1998). Subsequently it was shown thatthe deletion of four additional viral genes that are responsible forvirulence including, vpr, vif, vpu and nef did not alter the utility ofthe vector system (Zufferey et al., 1997). It was also shown that Tat,the main transactivator of HIV is also dispensable for the generation ofa fully efficient vector (Dull et al., 1998). Thus, the third-generationpackaging unit of the HIV-based lentiviral vectors comprise only threegenes of the parental virus: gag, pol and rev, which eliminates thepossibility of reconstitution of a wild-type virus throughrecombination.

This system was further improved by removing HIV transcriptional unitsfrom the vector (Zufferey et al., 1998). It was demonstrated thereinthat introducing a deletion in the U3 region of the 3′ LTR of the DNAused to produce the vector RNA generated self-inactivating (SIN)vectors. During reverse transcription this deletion is transferred tothe 5′ LTR of the proviral DNA. Enough sequence was eliminated,including the removal of a TATA box, which abolished the transcriptionalactivity of the LTR, which prevents production of full-length vector RNAin transduced cells. This however did not affect vector titers or the invitro or in vivo properties of the vector.

The present invention provides several improvements to the existinglentivectors as described above and in other parts of thisspecification. Introducing a lentivector providing a heterologous gene,such as genes to treat hematopoietic and lympho-hematopoietic disordersin this invention, into a packaging cell yields a producer cell whichreleases infectious viral particles carrying the foreign gene ofinterest.

The env gene can be derived from any virus, including retroviruses. Theenv preferably is an amphotropic envelope protein which allowstransduction of cells of human and other species. Examples ofretroviral-derived env genes include, but are not limited to: Moloneymurine leukemia virus (MoMuLV or MMLV), Harvey murine sarcoma virus(HaMuSV or HSV), murine mammary tumor virus (MuMTV or MMTV), gibbon apeleukemia virus (GaLV or GALV), human immunodeficiency virus (HIV) andRous sarcoma virus (RSV). Other env genes such as Vesicular stomatitisvirus (VSV) protein G (VSV G), that of hepatitis viruses and ofinfluenza also can be used.

While VSV G protein is a desirable env gene because VSV G confers broadhost range on the recombinant virus, VSV G can be deleterious to thehost cell. Thus, when a gene such as that for VSV G is used, it ispreferred to employ an inducible promoter system so that VSV Gexpression can be regulated to minimize host toxicity when VSV G isexpression is not required. For example, the tetracycline-regulatablegene expression system of Gossen & Bujard, (1992) can be employed toprovide for inducible expression of VSV G when tetracycline is withdrawnfrom the transferred cell. Thus, the tet/VP16 transactivator is presenton a first vector and the VSV G coding sequence is cloned downstreamfrom a promoter controlled by tet operator sequences on another vector.

The vector providing the viral env nucleic acid sequence is associatedoperably with regulatory sequences, e.g., a promoter or enhancer. Theregulatory sequence can be any eukaryotic promoter or enhancer,including for example, EF1α, PGK, the Moloney murine leukemia viruspromoter-enhancer element, the human cytomegalovirus enhancer, thevaccinia P7.5 promoter or the like (also see examples listed in Tables 1and 2 below). In some cases, such as the Moloney murine leukemia viruspromoter-enhancer element, the promoter-enhancer elements are locatedwithin or adjacent to the LTR sequences. Preferably, the regulatorysequence is one which is not endogenous to the lentivirus from which thevector is being constructed. Thus, if the vector is being made from SIV,the SIV regulatory sequence found in the SIV LTR would be replaced by aregulatory element which does not originate from SIV.

One may further target the recombinant virus by linkage of the envelopeprotein with an antibody or a particular ligand for targeting to areceptor of a particular cell-type. By inserting a sequence (including aregulatory region) of interest into the viral vector, along with anothergene which encodes the ligand for a receptor on a specific target cell,for example, the vector is now target-specific. Retroviral vectors canbe made target-specific by inserting, for example, a glycolipid or aprotein. Targeting often is accomplished by using an antigen-bindingportion of an antibody or a recombinant antibody-type molecule, such asa single chain antibody, to target the retroviral vector. Those of skillin the art will know of, or can readily ascertain without undueexperimentation, specific methods to achieve delivery of a retroviralvector to a specific target.

The heterologous or foreign nucleic acid sequence, such as apolynucleotide sequence encoding a gene such as a therapeutic gene forinherited or acquired hematopoietic disorders herein, is linked operablyto a regulatory nucleic acid sequence. Preferably, the heterologoussequence is linked to a promoter, resulting in a chimeric gene.

Marker genes may be utilized to assay for the presence of the vector,and thus, to confirm infection and integration. The presence of a markergene ensures the selection and growth of only those host cells whichexpress the inserts. Typical selection genes encode proteins that conferresistance to antibiotics and other toxic substances, e.g., histidinol,puromycin, hygromycin, neomycin, methotrexate, and cell surface markers.

The recombinant virus of the invention is capable of transferring anucleic acid sequence into a mammalian cell. The term, “nucleic acidsequence”, refers to any nucleic acid molecule, preferably DNA, asdiscussed in detail herein. The nucleic acid molecule may be derivedfrom a variety of sources, including DNA, cDNA, synthetic DNA, RNA orcombinations thereof. Such nucleic acid sequences may comprise genomicDNA which may or may not include naturally occurring introns. Moreover,such genomic DNA may be obtained in association with promoter regions,poly A sequences or other associated sequences. Genomic DNA may beextracted and purified from suitable cells by means well known in theart. Alternatively, messenger RNA (mRNA) can be isolated from cells andused to produce cDNA by reverse transcription or other means.

The vectors are introduced via transfection or infection into thepackaging cell line. The packaging cell line produces viral particlesthat contain the vector genome. Methods for transfection or infectionare well known by those of skill in the art. After cotransfection of thepackaging vectors and the transfer vector to the packaging cell line,the recombinant virus is recovered from the culture media and titteredby standard methods used by those of skill in the art. Thus, thepackaging constructs can be introduced into human cell lines by calciumphosphate transfection, lipofection or electroporation, generallytogether with a dominant selectable marker, such as neomycin, DHFR,Glutamine synthetase or ADA, followed by selection in the presence ofthe appropriate drug and isolation of clones. The selectable marker genecan be linked physically to the packaging genes in the construct.

Stable cell lines wherein the packaging functions are configured to beexpressed by a suitable packaging cell are known. For example, see U.S.Pat. No. 5,686,279; and Ory et al., (1996), which describe packagingcells. The packaging cells with a lentiviral vector incorporated in themform producer cells. Producer cells are thus cells or cell-lines thatcan produce or release packaged infectious viral particles carrying thetherapeutic gene of interest. These cells can further be anchoragedependent which means that these cells will grow, survive, or maintainfunction optimally when attached to a surface such as glass or plastic.The producer cells may also be neoplastically transformed cells. Someexamples of anchorage dependent cell lines used as lentiviral vectorpackaging cell lines when the vector is replication competent are HeLaor 293 cells and PERC.6 cells.

In some applications, particularly when the virus is to be used for genetherapy applications, it is preferable that the vector be replicationdeficient (or replication defective) to avoid uncontrolled proliferationof the virus in the individual to be treated. In such instancesmammalian cell lines are selected which have been engineered, either bymodification of the producer cell's genome to encode essential viralfunctions or by the co-infection of the producer cell with a helpervirus, to express proteins complementing the effect of the sequencesdeleted from the viral genome. For example, for HIV-1 derived vectors,the HIV-1 packaging cell line, PSI422, may be used as described inCorbeau, et al. (1996). Similarly, where the viral vector to be producedis a retrovirus, the human 293-derived retroviral packaging cell line(293GPG) capable of producing high titers of retroviral particles may beemployed as described in Ory, et al. (1996). In the production ofminimal vector systems, the producer cell is engineered (either bymodification of the viral genome or by the use of helper virus orcosmid) to complement the functions of the parent virus enablingreplication and packaging into virions in the producer cell line.

Lentiviral transfer vectors Naldini et al., (1996), have been used toinfect human cells growth-arrested in vitro and to transduce neuronsafter direct injection into the brain of adult rats. The vector wasefficient at transferring marker genes in vivo into the neurons and longterm expression in the absence of detectable pathology was achieved.Animals analyzed ten months after a single injection of the vectorshowed no decrease in the average level of transgene expression and nosign of tissue pathology or immune reaction (Blomer et al., (1997).

B. The Sin Design

The SIN design increases the biosaftey of the lentiviral vectors. Themajority of the HIV LTR is comprised of the U3 sequences. The U3 regioncontains the enhancer and promoter elements that modulate basal andinduced expression of the HIV genome in infected cells and in responseto cell activation. Several of these promoter elements are essential forviral replication. Some of the enhancer elements are highly conservedamong viral isolates and have been implicated as critical virulencefactors in viral pathogenesis. The enhancer elements may act toinfluence replication rates in the different cellular target of thevirus (Marthas et al., 1993).

As viral transcription starts at the 3′ end of the U3 region of the 5′LTR, those sequences are not part of the viral mRNA and a copy thereoffrom the 3′ LTR acts as template for the generation of both LTR's in theintegrated provirus. If the 3′ copy of the U3 region is altered in aretroviral vector construct, the vector RNA is still produced from theintact 5′ LTR in producer cells, but cannot be regenerated in targetcells. Transduction of such a vector results in the inactivation of bothLTR's in the progeny virus. Thus, the retrovirus is self-inactivating(SIN) and those vectors are known as SIN transfer vectors.

The SIN design is described in further detail in Zufferey et al., 1998and U.S. Pat. No. 5,994,136 both incorporated herein by reference. Asdescribed therein, there are, however, limits to the extent of thedeletion at the 3′ LTR. First, the 5′ end of the U3 region servesanother essential function in vector transfer, being required forintegration (terminal dinucleotide+att sequence). Thus, the terminaldinucleotide and the att sequence may represent the 5′ boundary of theU3 sequences which can be deleted. In addition, some loosely definedregions may influence the activity of the downstream polyadenylationsite in the R region. Excessive deletion of U3 sequence from the 3′ LTRmay decrease polyadenylation of vector transcripts with adverseconsequences both on the titer of the vector in producer cells and thetransgene expression in target cells. On the other hand, limiteddeletions may not abrogate the transcriptional activity of the LTR intransduced cells.

The lentiviral vectors described herein carry deletions of the U3 regionof the 3′ LTR spanning from nucleotide −418 to −18. This is the mostextensive deletion and extends as far as to the TATA box, thereforeabrogating any transcriptional activity of the LTR in transduced cells.The titer of vector in producer cells as well as transgene expression intarget cells was unaffected in these vectors. This design thereforeprovides an enormous increase in vector safety.

SIN-type vectors with such extensive deletions of the U3 region cannotbe generated for murine leukemia virus (MLV) or spleen necrosis virus(SNV) based retroviral vectors without compromising efficiency oftransduction.

Elimination of the −418 to −18 nucleotide sequence abolishestranscriptional activity of the LTR, thereby abolishing the productionof full length vector RNA in transduced cells. In the HIV-derivedlentivectors none of the in vitro or in vivo properties were compromisedby the SIN design.

C. Posttranscriptionally Regulating Elements (PRE)

Enhancing transgene expression may be required in certain embodiments,especially those that involve lentiviral constructs of the presentinvention with modest promoters.

One type of PRE is an intron positioned within the expression cassette,which can stimulate gene expression. However, introns can be spliced outduring the life cycle events of a lentivirus. Hence, if introns are usedas PRE's they have to be placed in an opposite orientation to the vectorgenomic transcript.

Posttranscriptional regulatory elements that do not rely on splicingevents offer the advantage of not being removed during the viral lifecycle. Some examples are the posttranscriptional processing element ofherpes simplex virus, the posttranscriptional regulatory element of thehepatitis B virus (HPRE) and the woodchuck hepatitis virus (WPRE). Ofthese the WPRE is most preferred as it contains an additional cis-actingelement not found in the HPRE (Donello et al., 1998). This regulatoryelement is positioned within the vector so as to be included in the RNAtranscript of the transgene, but outside of stop codon of the transgenetranslational unit. As demonstrated in the present invention and inZufferey et al., 1999, the WPRE element is a useful tool for stimulatingand enhancing gene expression of desired transgenes in the context ofthe lentiviral vectors.

The WPRE is characterized and described in U.S. Pat. No. 6,136,597,incorporated herein by reference. As described therein, the WPRE is anRNA export element that mediates efficient transport of RNA from thenucleus to the cytoplasm. It enhances the expression of transgenes byinsertion of a cis-acting nucleic acid sequence, such that the elementand the transgene are contained within a single transcript. Presence ofthe WPRE in the sense orientation was shown to increase transgeneexpression by up to 7 to 10 fold. Retroviral vectors transfer sequencesin the form of cDNAs instead of complete intron-containing genes asintrons are generally spliced out during the sequence of events leadingto the formation of the retroviral particle. Introns mediate theinteraction of primary transcripts with the splicing machinery. Becausethe processing of RNAs by the splicing machinery facilitates theircytoplasmic export, due to a coupling between the splicing and transportmachineries, cDNAs are often inefficiently expressed. Thus, theinclusion of the WPRE in a vector results in enhanced expression oftransgenes.

D. Nucleic Acids

One embodiment of the present invention is to transfer nucleic acidsencoding a therapeutic gene, especially a gene that provides therapy forhematopoietic and lympho-hematopoietic disorders, such as the inheritedor acquired disorders described above. In one embodiment the nucleicacids encode a full-length, substantially full-length, or functionalequivalent form of such a gene.

Thus, in some embodiments of the present invention, the treatment of ahematopoietic and lympho-hematopoietic disorder involves theadministration of a lentiviral vector of the invention comprising atherapeutic nucleic acid expression construct to a cell of hematopoieticorigin. It is contemplated that the hematopoietic cells take up theconstruct and express the therapeutic polypeptide encoded by nucleicacid, thereby restoring the cells normal phenotype.

A nucleic acid may be made by any technique known to one of ordinaryskill in the art. Non-limiting examples of synthetic nucleic acid,particularly a synthetic oligonucleotide, include a nucleic acid made byin vitro chemical synthesis using phosphotriester, phosphite orphosphoramidite chemistry and solid phase techniques such as describedin EP 266,032, incorporated herein by reference, or via deoxynucleosideH-phosphonate intermediates as described by Froehler et al., 1986, andU.S. Pat. No. 5,705,629, each incorporated herein by reference. Anon-limiting example of enzymatically produced nucleic acid include oneproduced by enzymes in amplification reactions such as PCR′ (see forexample, U.S. Pat. No. 4,683,202 and U.S. Pat. No. 4,682,195, eachincorporated herein by reference), or the synthesis of oligonucleotidesdescribed in U.S. Pat. No. 5,645,897, incorporated herein by reference.A non-limiting example of a biologically produced nucleic acid includesrecombinant nucleic acid production in living cells (see for example,Sambrook et al. 1989, incorporated herein by reference).

A nucleic acid may be purified on polyacrylamide gels, cesium chloridecentrifugation gradients, or by any other means known to one of ordinaryskill in the art (see for example, Sambrook et al. 1989, incorporatedherein by reference).

The term “nucleic acid” will generally refer to at least one molecule orstrand of DNA, RNA or a derivative or mimic thereof, comprising at leastone nucleobase, such as, for example, a naturally occurring purine orpyrimidine base found in DNA (e.g., adenine “A,” guanine “G,” thymine“T,” and cytosine “C”) or RNA (e.g. A, G, uracil “U,” and C). The term“nucleic acid” encompasses the terms “oligonucleotide” and“polynucleotide.” The term “oligonucleotide” refers to at least onemolecule of between about 3 and about 100 nucleobases in length. Theterm “polynucleotide” refers to at least one molecule of greater thanabout 100 nucleobases in length. These definitions generally refer to atleast one single-stranded molecule, but in specific embodiments willalso encompass at least one additional strand that is partially,substantially or fully complementary to the at least one single-strandedmolecule. Thus, a nucleic acid may encompass at least onedouble-stranded molecule or at least one triple-stranded molecule thatcomprises one or more complementary strand(s) or “complement(s)” of aparticular sequence comprising a strand of the molecule.

In certain embodiments, a “gene” refers to a nucleic acid that istranscribed. As used herein, a “gene segment” is a nucleic acid segmentof a gene. In certain aspects, the gene includes regulatory sequencesinvolved in transcription, or message production or composition. Inparticular embodiments, the gene comprises transcribed sequences thatencode for a protein, polypeptide or peptide. In other particularaspects, the gene comprises a nucleic acid, and/or encodes a polypeptideor peptide-coding sequences of a gene that is defective or mutated in ahematopoietic and lympho-hematopoietic disorder. In keeping with theterminology described herein, an “isolated gene” may comprisetranscribed nucleic acid(s), regulatory sequences, coding sequences, orthe like, isolated substantially away from other such sequences, such asother naturally occurring genes, regulatory sequences, polypeptide orpeptide encoding sequences, etc. In this respect, the term “gene” isused for simplicity to refer to a nucleic acid comprising a nucleotidesequence that is transcribed, and the complement thereof. In particularaspects, the transcribed nucleotide sequence comprises at least onefunctional protein, polypeptide and/or peptide encoding unit. As will beunderstood by those in the art, this functional term “gene” includesboth genomic sequences, RNA or cDNA sequences, or smaller engineerednucleic acid segments, including nucleic acid segments of anon-transcribed part of a gene, including but not limited to thenon-transcribed promoter or enhancer regions of a gene. Smallerengineered gene nucleic acid segments may express, or may be adapted toexpress using nucleic acid manipulation technology, proteins,polypeptides, domains, peptides, fusion proteins, mutants and/or suchlike. Thus, a “truncated gene” refers to a nucleic acid sequence that ismissing a stretch of contiguous nucleic acid residues.

Various nucleic acid segments may be designed based on a particularnucleic acid sequence, and may be of any length. By assigning numericvalues to a sequence, for example, the first residue is 1, the secondresidue is 2, etc., an algorithm defining all nucleic acid segments canbe created:n to n+ywhere n is an integer from 1 to the last number of the sequence and y isthe length of the nucleic acid segment minus one, where n+y does notexceed the last number of the sequence. Thus, for a 10-mer, the nucleicacid segments correspond to bases 1 to 10, 2 to 11, 3 to 12 . . . and/orso on. For a 15-mer, the nucleic acid segments correspond to bases 1 to15, 2 to 16, 3 to 17 . . . and/or so on. For a 20-mer, the nucleicsegments correspond to bases 1 to 20, 2 to 21, 3 to 22 . . . and/or soon.

The nucleic acid(s) of the present invention, regardless of the lengthof the sequence itself, may be combined with other nucleic acidsequences, including but not limited to, promoters, enhancers,polyadenylation signals, restriction enzyme sites, multiple cloningsites, coding segments, and the like, to create one or more nucleic acidconstruct(s). The overall length may vary considerably between nucleicacid constructs. Thus, a nucleic acid segment of almost any length maybe employed, with the total length preferably being limited by the easeof preparation or use in the intended recombinant nucleic acid protocol.

The term “vector” is used to refer to a carrier nucleic acid moleculeinto which a nucleic acid sequence can be inserted for introduction intoa cell where it can be replicated. Vectors of the present invention arelentivirus based as described above and in other parts of thespecification. The nucleic acid molecules carried by the vectors of theinvention encode therapeutic genes and will be used for carrying outgene-therapies. One of skill in the art would be well equipped toconstruct such a therapeutic vector through standard recombinanttechniques (see, for example, Maniatis et al., 1988 and Ausubel et al.,1994, both incorporated herein by reference).

The term “expression vector” refers to any type of genetic constructcomprising a nucleic acid coding for a RNA capable of being transcribed.In some cases, RNA molecules are then translated into a protein,polypeptide, or peptide. In other cases, these sequences are nottranslated, for example, in the production of antisense molecules orribozymes. Expression vectors can contain a variety of “controlsequences,” which refer to nucleic acid sequences necessary for thetranscription and possibly translation of an operably linked codingsequence in a particular host cell. In addition to control sequencesthat govern transcription and translation, vectors and expressionvectors may contain nucleic acid sequences that serve other functions aswell and are described below.

(a) Promoters and Enhancers

A “promoter” is a control sequence that is a region of a nucleic acidsequence at which initiation and rate of transcription are controlled.It may contain genetic elements at which regulatory proteins andmolecules may bind, such as RNA polymerase and other transcriptionfactors, to initiate the specific transcription a nucleic acid sequence.The phrases “operatively positioned,” “operatively linked,” “undercontrol,” and “under transcriptional control” mean that a promoter is ina correct functional location and/or orientation in relation to anucleic acid sequence to control transcriptional initiation and/orexpression of that sequence.

A promoter generally comprises a sequence that functions to position thestart site for RNA synthesis. The best known example of this is the TATAbox, but in some promoters lacking a TATA box, such as, for example, thepromoter for the mammalian terminal deoxynucleotidyl transferase geneand the promoter for the SV40 late genes, a discrete element overlyingthe start site itself helps to fix the place of initiation. Additionalpromoter elements regulate the frequency of transcriptional initiation.Typically, these are located in the region 30-110 bp upstream of thestart site, although a number of promoters have been shown to containfunctional elements downstream of the start site as well. To bring acoding sequence “under the control of” a promoter, one positions the 5′end of the transcription initiation site of the transcriptional readingframe “downstream” of (i.e., 3′ of) the chosen promoter. The “upstream”promoter stimulates transcription of the DNA and promotes expression ofthe encoded RNA.

The spacing between promoter elements frequently is flexible, so thatpromoter function is preserved when elements are inverted or movedrelative to one another. In the tk promoter, the spacing betweenpromoter elements can be increased to 50 bp apart before activity beginsto decline. Depending on the promoter, it appears that individualelements can function either cooperatively or independently to activatetranscription. A promoter may or may not be used in conjunction with an“enhancer,” which refers to a cis-acting regulatory sequence involved inthe transcriptional activation of a nucleic acid sequence.

A promoter may be one naturally associated with a nucleic acid sequence,as may be obtained by isolating the 5′ non-coding sequences locatedupstream of the coding segment and/or exon. Such a promoter can bereferred to as “endogenous.” Similarly, an enhancer may be one naturallyassociated with a nucleic acid sequence, located either downstream orupstream of that sequence. Alternatively, certain advantages will begained by positioning the coding nucleic acid segment under the controlof a recombinant or heterologous promoter, which refers to a promoterthat is not normally associated with a nucleic acid sequence in itsnatural environment. A recombinant or heterologous enhancer refers alsoto an enhancer not normally associated with a nucleic acid sequence inits natural environment. Such promoters or enhancers may includepromoters or enhancers of other genes, and promoters or enhancersisolated from any other virus, or prokaryotic or eukaryotic cell, andpromoters or enhancers not “naturally occurring,” i.e., containingdifferent elements of different transcriptional regulatory regions,and/or mutations that alter expression. For example, promoters that aremost commonly used in recombinant DNA construction include theβ-lactamase (penicillinase), lactose and tryptophan (trp) promotersystems. In addition to producing nucleic acid sequences of promotersand enhancers synthetically, sequences may be produced using recombinantcloning and/or nucleic acid amplification technology, including PCR™, inconnection with the compositions disclosed herein (see U.S. Pat. Nos.4,683,202 and 5,928,906, each incorporated herein by reference).Furthermore, it is contemplated the control sequences that directtranscription and/or expression of sequences within non-nuclearorganelles such as mitochondria, chloroplasts, and the like, can beemployed as well. Control sequences comprising promoters, enhancers andother locus or transcription controlling/modulating elements are alsoreferred to as “transcriptional cassettes”.

Naturally, it will be important to employ a promoter and/or enhancerthat effectively directs the expression of the DNA segment in theorganelle, cell type, tissue, organ, or organism chosen for expression.Those of skill in the art of molecular biology generally know the use ofpromoters, enhancers, and cell type combinations for protein expression,(see, for example Sambrook et al., 1989, incorporated herein byreference). The promoters employed may be constitutive, tissue-specific,inducible, and/or useful under the appropriate conditions to direct highlevel expression of the introduced DNA segment, such as is advantageousfor gene therapy or for applications such as the large-scale productionof recombinant proteins and/or peptides. The promoter may beheterologous or endogenous.

Use of a T3, T7 or SP6 cytoplasmic expression system is another possibleembodiment. Eukaryotic cells can support cytoplasmic transcription fromcertain bacterial promoters if the appropriate bacterial polymerase isprovided, either as part of the delivery complex or as an additionalgenetic expression construct.

Tables 1 lists non-limiting examples of elements/promoters that may beemployed, in the context of the present invention, to regulate theexpression of a RNA. Table 2 provides non-limiting examples of inducibleelements, which are regions of a nucleic acid sequence that can beactivated in response to a specific stimulus.

TABLE 1 Promoter and/or Enhancer Promoter/Enhancer ReferencesImmunoglobulin Heavy Chain Banerji et al., 1983; Gilles et al., 1983;Grosschedl et al, 1985; Atchinson et al., 1986, 1987; Imler et al.,1987; Weinberger et al., 1984; Kiledjian et al., 1988; Porton et al.;1990 Immunoglobulin Light Chain Queen et al, 1983; Picard et al., 1984T-Cell Receptor Luria et al., 1987; Winoto et al., 1989; Redondo et al.;1990 HLA DQ a and/or DQ β Sullivan et al., 1987 β-Interferon Goodbournet al., 1986; Fujita et al., 1987; Goodbourn et al., 1988 Interleukin-2Greene et al., 1989 Interleukin-2 Receptor Greene et al., 1989; Lin etal., 1990 MHC Class II 5 Koch et al., 1989 MHC Class II HLA-Dra Shermanet al., 1989 β-Actin Kawamoto et al., 1988; Ng et al.; 1989 MuscleCreatine Kinase (MCK) Jaynes et al., 1988; Horlick et al., 1989; Johnsonet al., 1989 Prealbumin (Transthyretin) Costa et al., 1988 Elastase IOmitz et al., 1987 Metallothionein (MTII) Karin et al, 1987; Culotta etal, 1989 Collagenase Pinkert et al., 1987; Angel et al., 1987 AlbuminPinkert et al., 1987; Tronche et al., 1989, 1990 α-Fetoprotein Godboutet al., 1988; Campere et al., 1989 γ-Globin Bodine et al., 1987;Perez-Stable et al., 1990 β-Globin Trudel et al., 1987 c-fos Cohen etal., 1987 c-HA-ras Triesman, 1986; Deschamps et al., 1985 Insulin Edlundet al., 1985 Neural Cell Adhesion Molecule Hirsh et al., 1990 (NCAM)α₁-Antitrypain Latimer et al., 1990 H2B (TH2B) Histone Hwang et al.,1990 Mouse and/or Type I Collagen Ripe et al., 1989 Glucose-RegulatedProteins Chang et al., 1989 (GRP94 and GRP78) Rat Growth Hormone Larsenet al., 1986 Human Serum Amyloid A (SAA) Edbrooke et al., 1989 TroponinI (TN I) Yutzey et al., 1989 Platelet-Derived Growth Factor Pech et al.,1989 (PDGF) Duchenne Muscular Dystrophy Klamut et al., 1990 SV40 Banerjiet al., 1981; Moreau et al., 1981; Sleigh et al., 1985; Firak et al.,1986; Herr et al., 1986; Imbra et al., 1986; Kadesch et al., 1986; Wanget al., 1986; Ondek et al., 1987; Kuhl et al., 1987; Schaffner et al.,1988 Polyoma Swartzendruber et al., 1975; Vasseur et al., 1980; Katinkaet al., 1980, 1981; Tyndell et al., 1981; Dandolo et al, 1983; deVilliers et al., 1984; Hen et al., 1986; Satake et al., 1988; Campbelland/or Villarreal, 1988 Retroviruses Kriegler et al., 1982, 1983;Levinson et al., 1982; Kriegler et al., 1983, 1984a, b, 1988; Bosze etal., 1986; Miksicek et al., 1986; Celander et al., 1987; Thiesen et al.,1988; Celander et al., 1988; Chol et al., 1988; Reisman et al., 1989Papilloma Virus Campo et al., 1983; Lusky et al., 1983; Spandidos and/orWilkie, 1983; Spalholz et al., 1985; Lusky et al., 1986; Cripe et al.,1987; Gloss et al., 1987; Hirochika et al., 1987; Stephens et al., 1987Hepatitis B Virus Bulla et al., 1986; Jameel et al., 1986; Shaul et al.,1987; Spandau et al., 1988; Vannice et al., 1988 Human ImmunodeficiencyVirus Muesing et al., 1987; Hauber et al., 1988; Jakobovits et al.,1988; Feng et al., 1988; Takebe et al., 1988; Rosen et al., 1988;Berkhout et al., 1989; Laspia et al., 1989; Sharp et al., 1989; Braddocket al., 1989 Gibbon Ape Leukemia Virus Holbrook et al., 1987; Quinn etal., 1989

TABLE 2 Inducible Elements Element Inducer References MT II PhorbolEster (TFA) Palmiter et al., 1982; Heavy metals Haslinger et al., 1985;Searle et al, 1985; Stuart et al., 1985; Imagawa et al., 1987, Karin etal., 1987; Angel et al., 1987b; McNeall et al., 1989 MMTV (mouseGlucocorticoids Huang et al., 1981; mammary Lee et al., 1981; tumorvirus) Majors et al., 1983; Chandler et al., 1983; Lee et al., 1984;Ponta et al., 1985; Sakai et al., 1988 β-Interferon Poly(rI)x Poly(rc)Tavernier et al., 1983 Adenovirus 5 E2 ElA Imperiale et al., 1984Collagenase Phorbol Ester (TPA) Angel et al., 1987a Stromelysin PhorbolEster (TPA) Angel et al., 1987b SV40 Phorbol Ester (TPA) Angel et al.,1987b Murine MX Gene Interferon, Newcastle Hug et al., 1988 DiseaseVirus GRP78 Gene A23187 Resendez et al., 1988 α-2-Macroglobulin IL-6Kunz et al., 1989 Vimentin Serum Rittling et al., 1989 MHC Class IInterferon Blanar et al., 1989 Gene H-2κb HSP70 ElA, SV40 Large T Tayloret al., 1989, Antigen 1990a, 1990b Proliferin Phorbol Ester-TPA Mordacqet al., 1989 Tumor Necrosis Factor PMA Hensel et al., 1989 ThyroidStimulating Thyroid Hormone Chatterjee et al., 1989 Hormone α Gene

The identity of tissue-specific promoters or elements, as well as assaysto characterize their activity, is well known to those of skill in theart. Non-limiting examples of such regions include the human LIMK2 gene(Nomoto et al., 1999), the somatostatin receptor 2 gene (Kraus et al.,1998), murine epididymal retinoic acid-binding gene (Lareyre et al.,1999), human CD4 (Zhao-Emonet et al., 1998), mouse alpha2 (XI) collagen(Tsumaki, et al., 1998), D1A dopamine receptor gene (Lee, et al., 1997),insulin-like growth factor II (Wu et al., 1997), and human plateletendothelial cell adhesion molecule-1 (Almendro et al., 1996).

The lentiviral vectors of the present invention are designed, primarily,to transform cells with a therapeutic gene under the control ofregulated eukaryotic promoters. Although the EF1α-promoter and the PGKpromoter are preferred other promoter and regulatory signal elements asdescribed in the Tables 1 and 2 above may also be used. Additionally anypromoter/enhancer combination (as per the Eukaryotic Promoter Data BaseEPDB) could also be used to drive expression of structural genesencoding the therapeutic gene of interest that is used in context withthe lentiviral vectors of the present invention. Alternatively, atissue-specific promoter for cancer gene therapy or the targeting oftumors may be employed with the lentiviral vectors of the presentinvention for treatment of cancers, especially hematological cancers.

Typically promoters and enhancers that control the transcription ofprotein encoding genes in eukaryotic cells are composed of multiplegenetic elements. The cellular machinery is able to gather and integratethe regulatory information conveyed by each element, allowing differentgenes to evolve distinct, often complex patterns of transcriptionalregulation.

Enhancers were originally detected as genetic elements that increasedtranscription from a promoter located at a distant position on the samemolecule of DNA. This ability to act over a large distance had littleprecedent in classic studies of prokaryotic transcriptional regulation.Subsequent work showed that regions of DNA with enhancer activity areorganized much like promoters. That is, they are composed of manyindividual elements, each of which binds to one or more transcriptionalproteins.

The basic distinction between enhancers and promoters is operational. Anenhancer region as a whole must be able to stimulate transcription at adistance; this need not be true of a promoter region or its componentelements. On the other hand, a promoter must have one or more elementsthat direct initiation of RNA synthesis at a particular site and in aparticular orientation, whereas enhancers lack these specificities.Aside from this operational distinction, enhancers and promoters arevery similar entities.

Promoters and enhancers have the same general function of activatingtranscription in the cell. They are often overlapping and contiguous,often seeming to have a very similar modular organization. Takentogether, these considerations suggest that enhancers and promoters arehomologous entities and that the transcriptional activator proteinsbound to these sequences may interact with the cellular transcriptionalmachinery in fundamentally the same way.

A signal that may prove useful is a polyadenylation signal (hGH, BGH,SV40). The use of internal ribosome binding sites (IRES) elements areused to create multigene, or polycistronic, messages. IRES elements areable to bypass the ribosome scanning model of 5′-methylatedcap-dependent translation and begin translation at internal sites(Pelletier and Sonenberg, 1988). IRES elements from two members of thepicornavirus family (polio and encephalomyocarditis) have been described(Pelletier and Sonenberg, 1988), as well as an IRES from a mammalianmessage (Macejak and Sarnow, 1991). IRES elements can be linked toheterologous open reading frames. Multiple open reading frames can betranscribed together, each separated by an IRES, creating polycistronicmessages. By virtue of the IRES element, each open reading frame isaccessible to ribosomes for efficient translation. Multiple genes can beefficiently expressed using a single promoter/enhancer to transcribe asingle message.

In any event, it will be understood that promoters are DNA elementswhich when positioned functionally upstream of a gene leads to theexpression of that gene. Most transgenes that will be transformed usingthe lentiviral vectors of the present invention are functionallypositioned downstream of a promoter element.

A specific initiation signal also may be required for efficienttranslation of coding sequences. These signals include the ATGinitiation codon or adjacent sequences. Exogenous translational controlsignals, including the ATG initiation codon, may need to be provided.One of ordinary skill in the art would readily be capable of determiningthis and providing the necessary signals. It is well known that theinitiation codon must be “in-frame” with the reading frame of thedesired coding sequence to ensure translation of the entire insert. Theexogenous translational control signals and initiation codons can beeither natural or synthetic. The efficiency of expression may beenhanced by the inclusion of appropriate transcription enhancerelements.

(b) Multiple Cloning Sites

Vectors of the present invention can include a multiple cloning site(MCS), which is a nucleic acid region that contains multiple restrictionenzyme sites, any of which can be used in conjunction with standardrecombinant technology to digest the vector (see, for example,Carbonelli et al., 1999, Levenson et al., 1998, and Cocea, 1997,incorporated herein by reference.) “Restriction enzyme digestion” refersto catalytic cleavage of a nucleic acid molecule with an enzyme thatfunctions only at specific locations in a nucleic acid molecule. Many ofthese restriction enzymes are commercially available. Use of suchenzymes is widely understood by those of skill in the art. Frequently, avector is linearized or fragmented using a restriction enzyme that cutswithin the MCS to enable exogenous sequences to be ligated to thevector. “Ligation” refers to the process of forming phosphodiester bondsbetween two nucleic acid fragments, which may or may not be contiguouswith each other. Techniques involving restriction enzymes and ligationreactions are well known to those of skill in the art of recombinanttechnology.

(c) Splicing Sites

Most transcribed eukaryotic RNA molecules will undergo RNA splicing toremove introns from the primary transcripts. Vectors containing genomiceukaryotic sequences may require donor and/or acceptor splicing sites toensure proper processing of the transcript for protein expression (see,for example, Chandler et al., 1997, herein incorporated by reference.)

(d) Termination Signals

The vectors or constructs of the present invention will generallycomprise at least one termination signal. A “termination signal” or“terminator” is comprised of the DNA sequences involved in specifictermination of an RNA transcript by an RNA polymerase. Thus, in certainembodiments a termination signal that ends the production of an RNAtranscript is contemplated. A terminator may be necessary in vivo toachieve desirable message levels.

In eukaryotic systems, the terminator region may also comprise specificDNA sequences that permit site-specific cleavage of the new transcriptso as to expose a polyadenylation site. This signals a specializedendogenous polymerase to add a stretch of about 200 A residues (polyA)to the 3′ end of the transcript. RNA molecules modified with this polyAtail appear to more stable and are translated more efficiently. Thus, inother embodiments involving eukaryotes, it is preferred that thatterminator comprises a signal for the cleavage of the RNA, and it ismore preferred that the terminator signal promotes polyadenylation ofthe message. The terminator and/or polyadenylation site elements canserve to enhance message levels and to minimize read through from thecassette into other sequences.

Terminators contemplated for use in the invention include any knownterminator of transcription described herein or known to one of ordinaryskill in the art, including but not limited to, for example, thetermination sequences of genes, such as for example the bovine growthhormone terminator or viral termination sequences, such as for examplethe SV40 terminator. In certain embodiments, the termination signal maybe a lack of transcribable or translatable sequence, such as due to asequence truncation.

(e) Polyadenylation Signals

In eukaryotic gene expression, one will typically include apolyadenylation signal to effect proper polyadenylation of thetranscript. The nature of the polyadenylation signal is not believed tobe crucial to the successful practice of the invention, and any suchsequence may be employed. Some examples include the SV40 polyadenylationsignal or the bovine growth hormone polyadenylation signal, convenientand known to function well in various target cells. Polyadenylation mayincrease the stability of the transcript or may facilitate cytoplasmictransport.

(f) Origins of Replication

In order to propagate a vector of the invention in a host cell, it maycontain one or more origins of replication sites (often termed “ori”),which is a specific nucleic acid sequence at which replication isinitiated. Alternatively an autonomously replicating sequence (ARS) canbe employed if the host cell is yeast.

(g) Selectable and Screenable Markers

In certain embodiments of the invention, cells transduced with thelentivectors of the present invention may be identified in vitro or invivo by including a marker in the expression vector. Such markers wouldconfer an identifiable change to the transduced cell permitting easyidentification of cells containing the expression vector. Generally, aselectable marker is one that confers a property that allows forselection. A positive selectable marker is one in which the presence ofthe marker allows for its selection, while a negative selectable markeris one in which its presence prevents its selection. An example of apositive selectable marker is a drug resistance marker.

Usually the inclusion of a drug selection marker aids in the cloning andidentification of transformants, for example, genetic constructs thatconfer resistance to neomycin, puromycin, hygromycin, DHFR, GPT, zeocinand histidinol are useful selectable markers. In addition to markersconferring a phenotype that allows for the discrimination oftransformants based on the implementation of conditions, other types ofmarkers including screenable markers such as GFP, whose basis iscolorimetric analysis, are also contemplated. Alternatively, screenableenzymes such as herpes simplex virus thymidine kinase (tk) orchloramphenicol acetyltransferase (CAT) may be utilized. One of skill inthe art would also know how to employ immunologic markers, possibly inconjunction with FACS analysis. The marker used is not believed to beimportant, so long as it is capable of being expressed simultaneouslywith the nucleic acid encoding a gene product. Further examples ofselectable and screenable markers are well known to one of skill in theart.

E. Host Cells

As used herein, the terms “cell,” “cell line,” and “cell culture” may beused interchangeably. All of these terms also include their progeny,which is any and all subsequent generations. It is understood that allprogeny may not be identical due to deliberate or inadvertent mutations.In the context of expressing a heterologous nucleic acid sequence, “hostcell” refers to a prokaryotic or eukaryotic cell, and it includes anytransformable organisms that is capable of replicating a vector and/orexpressing a heterologous nucleic acid encoded by the vectors of thisinvention. A host cell can, and has been, used as a recipient forvectors. A host cell may be “transfected” or “transformed,” which refersto a process by which exogenous nucleic acid is transferred orintroduced into the host cell. A transformed cell includes the primarysubject cell and its progeny. As used herein, the terms “engineered” and“recombinant” cells or host cells are intended to refer to a cell intowhich an exogenous nucleic acid sequence, such as, for example, alentivector of the invention bearing a therapeutic gene construct, hasbeen introduced. Therefore, recombinant cells are distinguishable fromnaturally occurring cells which do not contain a recombinantlyintroduced nucleic acid.

In certain embodiments, it is contemplated that RNAs or proteinaceoussequences may be co-expressed with other selected RNAs or proteinaceoussequences in the same host cell. Co-expression may be achieved byco-transfecting the host cell with two or more distinct recombinantvectors. Alternatively, a single recombinant vector may be constructedto include multiple distinct coding regions for RNAs, which could thenbe expressed in host cells transfected with the single vector.

Host cells may be derived from prokaryotes or eukaryotes, depending uponwhether the desired result is replication of the vector or expression ofpart or all of the vector-encoded nucleic acid sequences. Numerous celllines and cultures are available for use as a host cell, and they can beobtained through the American Type Culture Collection (ATCC), which isan organization that serves as an archive for living cultures andgenetic materials (www.atcc.org). Some examples of host cells used inthis invention include but are not limited to virus packaging cells,virus producer cells, 293T cells, human hematopoietic progenitor cells,human hematopoietic stem cells, CD34⁺ cells CD4⁺ cells, and the like.

(a) Tissues and Cells

A tissue may comprise a host cell or cells to be transformed orcontacted with a nucleic acid delivery composition and/or an additionalagent. The tissue may be part or separated from an organism. In certainembodiments, a tissue and its constituent cells may comprise, but is notlimited to, blood (e.g., hematopoietic cells (such as humanhematopoietic progenitor cells, human hematopoietic stem cells, CD34⁺cells CD4⁺ cells), lymphocytes and other blood lineage cells), bonemarrow, brain, stem cells, blood vessel, liver, lung, bone, breast,cartilage, cervix, colon, cornea, embryonic, endometrium, endothelial,epithelial, esophagus, facia, fibroblast, follicular, ganglion cells,glial cells, goblet cells, kidney, lymph node, muscle, neuron, ovaries,pancreas, peripheral blood, prostate, skin, skin, small intestine,spleen, stomach, testes.

(b) Organisms

In certain embodiments, the host cell or tissue may be comprised in atleast one organism. In certain embodiments, the organism may be, human,primate or murine. In other embodiments the organism may be anyeukaryote or even a prokayote (e.g., a eubacteria, an archaea), as wouldbe understood by one of ordinary skill in the art (see, for example,webpage http://phylogeny.arizona.edu/tree/phylogeny.html). Somelentivectors of the invention may employ control sequences that allowthem to be replicated and/or expressed in both prokaryotic andeukaryotic cells. One of skill in the art would further understand theconditions under which to incubate all of the above described host cellsto maintain them and to permit replication of a vector. Also understoodand known are techniques and conditions that would allow large-scaleproduction of the lentivectors of the invention, as well as productionof the nucleic acids encoded by the lentivectors and their cognatepolypeptides, proteins, or peptides some of which are therapeutic genesor proteins which will be used for gene therapies.

F. Injectable Compositions and Pharmaceutical Formulations

To achieve gene-therapy using the lentiviral vector compositions of thepresent invention, one would generally contact a cell in need thereofwith a lentiviral vector comprising a therapeutic gene. The cell willfurther be in an organism such as a human in need of the gene therapy.The routes of administration will vary, naturally, with the location andnature of the disease, and include, e.g., intravenous, intrarterial,intradermal, transdermal, intramuscular, intranasal, subcutaneous,percutaneous, intratracheal, intraperitoneal, intratumoral, perfusionand lavage. The cells will also sometimes be isolated from theorganisms, exposed to the lentivector ex vivo, and reimplantedafterwards.

Injection of lentiviral nucleic acid constructs of the invention may bedelivered by syringe or any other method used for injection of asolution, as long as the expression construct can pass through theparticular gauge of needle required for injection. A novel needlelessinjection system has recently been described (U.S. Pat. No. 5,846,233)having a nozzle defining an ampule chamber for holding the solution andan energy device for pushing the solution out of the nozzle to the siteof delivery. A syringe system has also been described for use in genetherapy that permits multiple injections of predetermined quantities ofa solution precisely at any depth (U.S. Pat. No. 5,846,225).

Solutions of the nucleic acids as free base or pharmacologicallyacceptable salts may be prepared in water suitably mixed with asurfactant, such as hydroxypropylcellulose. Dispersions may also beprepared in glycerol, liquid polyethylene glycols, and mixtures thereofand in oils. Under ordinary conditions of storage and use, thesepreparations contain a preservative to prevent the growth ofmicroorganisms. The pharmaceutical forms suitable for injectable useinclude sterile aqueous solutions or dispersions and sterile powders forthe extemporaneous preparation of sterile injectable solutions ordispersions (U.S. Pat. No. 5,466,468, specifically incorporated hereinby reference in its entirety). In all cases the form must be sterile andmust be fluid to the extent that easy syringability exists. It must bestable under the conditions of manufacture and storage and must bepreserved against the contaminating action of microorganisms, such asbacteria and fungi. The carrier can be a solvent or dispersion mediumcontaining, for example, water, ethanol, polyol (e.g., glycerol,propylene glycol, and liquid polyethylene glycol, and the like),suitable mixtures thereof, and/or vegetable oils. Proper fluidity may bemaintained, for example, by the use of a coating, such as lecithin, bythe maintenance of the required particle size in the case of dispersionand by the use of surfactants. The prevention of the action ofmicroorganisms can be brought about by various antibacterial andantifungal agents, for example, parabens, chlorobutanol, phenol, sorbicacid, thimerosal, and the like. In many cases, it will be preferable toinclude isotonic agents, for example, sugars or sodium chloride.Prolonged absorption of the injectable compositions can be brought aboutby the use in the compositions of agents delaying absorption, forexample, aluminum monostearate and gelatin.

For parenteral administration in an aqueous solution, for example, thesolution should be suitably buffered if necessary and the liquid diluentfirst rendered isotonic with sufficient saline or glucose. Theseparticular aqueous solutions are especially suitable for intravenous,intraarterial, intramuscular, subcutaneous, intratumoral andintraperitoneal administration. In this connection, sterile aqueousmedia that can be employed will be known to those of skill in the art inlight of the present disclosure. For example, one dosage may bedissolved in 1 ml of isotonic NaCl solution and either added to 1000 mlof hypodermoclysis fluid or injected at the proposed site of infusion,(see for example, “Remington's Pharmaceutical Sciences” 15th Edition,pages 1035-1038 and 1570-1580). Some variation in dosage willnecessarily occur depending on the condition of the subject beingtreated. The person responsible for administration will, in any event,determine the appropriate dose for the individual subject. Moreover, forhuman administration, preparations should meet sterility, pyrogenicity,general safety and purity standards as required by FDA Office ofBiologics standards.

Sterile injectable solutions are prepared by incorporating the activecompounds in the required amount in the appropriate solvent with variousof the other ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the various sterilized active ingredients into a sterilevehicle which contains the basic dispersion medium and the requiredother ingredients from those enumerated above. In the case of sterilepowders for the preparation of sterile injectable solutions, thepreferred methods of preparation are vacuum-drying and freeze-dryingtechniques which yield a powder of the active ingredient plus anyadditional desired ingredient from a previously sterile-filteredsolution thereof.

The compositions disclosed herein may be formulated in a neutral or saltform. Pharmaceutically-acceptable salts, include the acid addition saltsand which are formed with inorganic acids such as, for example,hydrochloric or phosphoric acids, or such organic acids as acetic,oxalic, tartaric, mandelic, and the like. Salts formed with the freecarboxyl groups can also be derived from inorganic bases such as, forexample, sodium, potassium, ammonium, calcium, or ferric hydroxides, andsuch organic bases as isopropylamine, trimethylamine, histidine,procaine and the like. Upon formulation, solutions will be administeredin a manner compatible with the dosage formulation and in such amount asis therapeutically effective. The formulations are easily administeredin a variety of dosage forms such as injectable solutions, drug releasecapsules and the like.

As used herein, “carrier” includes any and all solvents, dispersionmedia, vehicles, coatings, diluents, antibacterial and antifungalagents, isotonic and absorption delaying agents, buffers, carriersolutions, suspensions, colloids, and the like. The use of such mediaand agents for pharmaceutical active substances is well known in theart. Except insofar as any conventional media or agent is incompatiblewith the active ingredient, its use in the therapeutic compositions iscontemplated. Supplementary active ingredients can also be incorporatedinto the compositions.

The phrase “pharmaceutically-acceptable” or“pharmacologically-acceptable” refers to molecular entities andcompositions that do not produce an allergic or similar untowardreaction when administered to a human. The preparation of an aqueouscomposition that contains a protein as an active ingredient is wellunderstood in the art. Typically, such compositions are prepared asinjectables, either as liquid solutions or suspensions; solid formssuitable for solution in, or suspension in, liquid prior to injectioncan also be prepared.

The terms “contacted” and “exposed,” when applied to a cell, are usedherein to describe the process by which a therapeutic lentiviral vectoris delivered to a target cell.

For gene-therapy to discrete, solid, accessible tumors, intratumoralinjection, or injection into the tumor vasculature is specificallycontemplated. Local, regional or systemic administration also may beappropriate. For tumors of >4 cm, the volume to be administered will beabout 4-10 ml (preferably 10 ml), while for tumors of <4 cm, a volume ofabout 1-3 ml will be used (preferably 3 ml). Multiple injectionsdelivered as single dose comprise about 0.1 to about 0.5 ml volumes. Theviral particles may advantageously be contacted by administeringmultiple injections to the tumor, spaced at approximately 1 cmintervals. Systemic administration is preferred for conditions such ashematological malignancies.

Continuous administration also may be applied where appropriate.Delivery via syringe or catherization is preferred. Such continuousperfusion may take place for a period from about 1-2 hours, to about 2-6hours, to about 6-12 hours, to about 12-24 hours, to about 1-2 days, toabout 1-2 wk or longer following the initiation of treatment. Generally,the dose of the therapeutic composition via continuous perfusion will beequivalent to that given by a single or multiple injections, adjustedover a period of time during which the perfusion occurs.

Treatment regimens may vary as well, and often depend on type of diseaseand location of diseased tissue, and factors such as the health and theage of the patient. The clinician will be best suited to make suchdecisions based on the known efficacy and toxicity (if any) of thetherapeutic formulations based on lentiviral vectors of the presentinvention.

The treatments may include various “unit doses.” A unit dose is definedas containing a predetermined-quantity of the therapeutic compositioncomprising a lentiviral vector of the present invention. The quantity tobe administered, and the particular route and formulation, are withinthe skill of those in the clinical arts. A unit dose need not beadministered as a single injection but may comprise continuous infusionover a set period of time. Unit dose of the present invention mayconveniently be described in terms of transducing units (T.U.) oflentivector, as defined by tittering the vector on a cell line such asHeLa or 293. Unit doses range from 10³, 10⁴, 10⁵, 10⁶, 10⁷, 10⁸, 10⁹,10¹⁰, 10¹¹, 10¹², 10¹³ T.U. and higher.

G. Examples

The following examples are included to demonstrate preferred embodimentsof the invention. It should be appreciated by those of skill in the artthat the techniques disclosed in the examples which follow representtechniques discovered by the inventor to function well in the practiceof the invention, and thus can be considered to constitute preferredmodes for its practice. However, those of skill in the art should, inlight of the present disclosure, appreciate that many changes can bemade in the specific embodiments which are disclosed and still obtain alike or similar result without departing from the spirit and scope ofthe invention.

Materials and Methodology Employed in Examples 1 Through 5

Vector Preparation

Production of MLV- and HIV-derived vectors pseudotyped with thevesicular stomatitis virus (VSV) G envelope protein was achieved bytransient co-transfection of three plasmids into 293T epithelial cellline as described in Naldini et al 1996a. MLV vector particles wereproduced using the CMV-GagPol plasmid as packaging construct (Scharfmannet al., 1991), and a vector derived from pSLX, which expressed GFP fromthe CMV promoter. The HIV-derived packaging construct used waspCMVAR8.91, which encodes the HIV-1 Gag and Pol precursors, as well asthe regulatory proteins Tat and Rev (Zufferey et al., 1997). VSV G wasexpressed from pMD.G.

The HIV vector plasmids were derivatives of the original pHR′ backbone(Naldini et al., 1996a), with the following modifications.Self-inactivating vectors were produced from the previously describedSIN-18 vector, which contains a deletion in the U3 region of the 3′LTRfrom nt. −418 to nt. −18, removing all the transcriptionally activesequences (Zufferey et al., 1998). Briefly, the pHR′ SIN plasmids (alsosee maps in FIG. 6, FIG. 7, FIG. 9 and FIG. 9) were generated asfollows; a KpnI-XbaI fragment containing the polypurine tract and the3′LTR was excised from a pHR′ plasmid and subcloned into thecorresponding sites of pUC18. This plasmid was digested completely withEcoRV and partially with PvuII and self-ligated. A plasmid carrying a400-nucleotide deletion of U3 was recovered. An XhoI linker was insertedinto the EcoRI site of the deletion plasmid, and an XhoI-XbaI fragmentwas cloned back into the pHR′ CMVlacZ plasmid digested with thecorresponding enzymes. All other SIN-18 plasmids were obtained bysubstituting reporter genes (encoding luciferase, GFP and Neo) for lacZ.The pHR′ vector plasmids used differed from the plasmids originallydescribed (Naldini et al., 1996) by a XhoI-KpnI deletion removing 118nucleotides from the Nef coding sequence upstream of the polypurinetract and a deletion of 1,456 nucleotides of human sequence downstreamof the 3′ LTR. This human sequence remained from the original cloning ofa HXB2 proviral genome. The two deletions did not affect vector titersor transgene expression in dividing 293T cells.

Insertion of the EF1alpha was done by inserting a ClaI-BamHI cassettecontaining a EF1-GFP insert, into ClaI-BamHI site of pHR′-GFP-W-SIN(Zufferey et al. 1999)

The maps shown in FIG. 6, FIG. 7, FIG. 9 and FIG. 9 depict the plasmidconstructs pHR-CMV-GFP, pHR-EF1-GFP, pHR-EF1-GFP-SIN andpHR-EF1-GFP-W-SIN.

The phosphoglycerate kinase (PGK) promoter corresponded to nucleotides424 to 930 of the murine PGK gene (Genbank accession number M18735). TheEF1α promoter was derived from the pEF-BOS plasmid (Mizushima andNagata, 1990), and comprised nucleotides 373 to 1561 of the humanelongation factor EF1α, gene (Genbank accession number: J04617). Aftertransient transfection of the three plasmids by calcium phosphate in293T cells, the supernatant was harvested, concentrated by 2 rounds ofultracentrifugation, and resuspended in serum-free Cellgro® SCGM medium(BioWhittaker Europe, Verviers, Belgium). Viral stocks were stored at−70° C. and titers determined by transduction and flow cytometryanalysis of GFP expression in HeLa cells as previously described(Zufferey et al., 1997). Titers were comprised between 5×10⁷ and 10⁸HeLa-transducing units (TU) per ml.

Purification and Transduction of CD34⁺ Cells

Cord blood (CB) samples were obtained according to institutionalguidelines and CD34⁺ cells were purified as described (Arrighi et al.,1999). In brief, CB mononuclear cells recovered after Ficoll-Paque(Pharmacia, Uppsala, Sweden) gradient centrifugation were incubated onice with anti-CD34 M450 Dynabeads as described by the manufacturer.After several washes to eliminate unbound cells, CD34⁺ cells wererecovered from the beads by incubation for 15 minutes at 37° C. with the“Detach-a-bead” included in the kit. Cells were immediately washed, andanalyzed by flow cytometry. The percentage of purified CD34⁺ cells was89±7.0. For transduction, 10⁵ cells were seeded in 96-well plates in 100μl of Cellgro® SCGM medium supplemented with antibiotics (Gibco BRL,Life Technologies LTD, Paisley, Scotland, U.K.), with 10⁴ Mdithiothreitol (Fluka Biochemika, Buchs, Switzerland) and TPO. Afterovernight incubation, 10⁶ (typically), or 10⁵ to 5×10⁶ (fordose-response analysis) HeLa-transducing units (TU) of vector were addedper well, and the volume was adjusted to 200 μl with Cellgro® SCGMmedium containing TPO. After 24 hours, cells were washed, diluted to 400μl in IMDM supplemented with 10% of FCS (both from Gibco BRL, LifeTechnologies LTD), antibiotics, and FLT3-L, TPO and SCF for 3 days.Cells were either directly analyzed for GFP and CD34 expression, orfurther cultured with the 3 growth factors. For experiments shown inFIG. 1, concentrated viral stocks were treated with DNAse I prior toincubation with cells in order to eliminate DNA contaminants that mayinterfere with further PCR analysis. Briefly, 10⁶ TU of vector wasincubated for 30′ at 37° C. in a final volume of 20 μl containing 20μg/ml Dnase I (DNAse I, Roche Diagnostics, Rotkreuz, Switzerland) and 10mM MgCl2. Then, 80 μl of Cellgro was added and the resulting 100 μl wereadded to 100 μl of Cellgro containing 10⁵ CD34+ cells. Cells were thencultured as described above.

PCR Analysis after Transduction of CD34⁺ Cells

Cells (5×10⁵) were lysed in 18 μl of 50 mM Tris.HCl, pH 8.0, 20 mM NaCl,1 mM EDTA, 1% SDS. Proteinase K (2 μl at 10 mg/ml) was added and sampleswere incubated for 30′ at 55° C. After digestion, 200 μl of water wasadded and samples were boiled for 10′ to inactivate proteinase K. PCRamplification was performed using HotStarTaq (Qiagen, GmbH, Hilden,Germany) in a final volume of 50 μl and 2 μl or 0.7 μl of extract withthe following cycles: 95° C. 15′; 40 cycles 94° C. 30″, 60° C. 1′, 72°C. 1′, plus 10′ extension phase at 72° C. Sequences of the PCR primerswere: 5′ hIL2 gcaactcctgtcttgcattg; 3′ hIL2 aatgtgagcatcctggtgag; 5′ GFPgtgagcaagggcgaggagc; 3′GFP cttgatgccgttcttctgcttgt. Conditions wereadjusted to observe decrease in final PCR product yield when dilutingsamples by a factor of 3 (see FIG. 1A).

Purification and Transduction of Primary Human T Cells

Peripheral blood mononuclear cells were purified from buffy coats ofhealthy donors over a Ficoll-Hypaque gradient. Macrophages were removedby plastic adherence for 2 hrs at 37° C., and the remaining cells wereincubated with a cocktail of monoclonal antibodies against HLA-DR, CD25,CD69, CD19, CD16, CD11b and CD14 (Pharmingen). After a 30-min incubationon ice, cells were washed twice and incubated with magnetic beads(Dynabeads; Dynal, Oslo, Norway) conjugated with goat anti-mouse IgG, ata 1:4 target/bead ratio. Thirty minutes later, bead-bound cells wereremoved using a magnet. Remaining cells were further purified throughtwo more rounds at an increased target/bead ratio (1:10). Thispurification protocol typically resulted in a 99.5% pure population ofresting T cells, as determined by flow cytometry analysis withantibodies against the activation markers HLA-DR, CD25 and CD69. Cellsmaintained in RPMI 1640 supplemented with 10% FCS, 5 mM penicillin andstreptomycin (GIBCO BRL) and 2 mM glutamine (GIBCO BRL) were activatedwith phytohemagglutinin (Sigma) at 3 μg/ml for 48 hrs and subsequentlycultured in RPMI 1640 containing 10% fetal calf serum and recombinantinterleukin-2 (Sigma) at 10 U/ml. 5×10⁵ cells were transduced withvarious lentiviral vectors at a MOI of 5 HeLa-transducing unit per cellin 24-well plates in the presence of PHA, in a final volume of 500 μl.Flow cytometry analysis of GFP expression was performed five days aftertransduction.

Cytokines

All cytokines were recombinant human material. GM-CSF (Leucomax) fromEssex Chimie & Sandoz (Basel, Switzerland) was used at 20 ng/ml, G-CSF(Neupogen) from Roche (Basel, Switzerland) at 10 ng/ml, anderythropoietin (Eprex) from Cilag (Schaffhausen, Switzerland), at 2U/ml. Other cytokines were purchased from Peprotech EC (London, U.K.)and used at the following concentrations: FLT3-L 25 ng/ml, TPO 10 U/ml,SCF 20 ng/ml, interleukin-3 10 ng/ml, TNF 40 ng/ml and IL-4 20 ng/ml.

Antibodies and Immunoreactants

Phycoerythrin-conjugated monoclonal antibodies (MAb) were the following:anti-CD34 (mIgG1 clone 8G12) from Becton-Dickinson (Mountain View,Calif.), anti-glycophorin-A (mIgG1, clone JC 159), anti-CD42b (mIgG2a,clone AN51) an isotypic controls from Dako A/S (Glostrup, Denmark).Biotin-labeled MAbs: Anti-CD14 (mIgG2a, clone UCHM1) from Ancell Corp(Bayport, Minn.), and anti-CD15 (mIgM cloneDU-HL60-3) from Sigma (StLouis, Mo.), and isotypic controls from Ancell. Allophycocyanin(APC)-labeled streptavidin was from Pharmingen (San Diego, Calif.).Anti-CD34 mIgG coated M450 Dynabeads were from Dynal A/S (Oslo, Norway).

In Vitro Differentiation

Megakaryocytic differentiation was evaluated after 10 days of expansionculture with FLT3-L, TPO, and SCF. For the other lineages, cells werewashed after 9-11 days of expansion culture with FLT3-L, TPO, and SCF,and incubated with EPO, IL-3 and SCF for erythroid differentiation,GM-CSF and SCF for monocytic differentiation, and G-CSF and SCF forgranulocytic differentiation. Differentiation into dendritic cells wasperformed as previously described (Arrighi et al., 1999). Briefly,transduced CD34+ cells were expanded for 14 to 28 days with FLT3-L, TPO,and SCF. Cells were then induced into mature DC by exposure to GM-CSFand IL-4 for 3 days, followed by GM-CSF, IL-4 and TNF for 3 more days.The amplification ratio was comprised between 100 and 1000 depending onthe experiment and the cell lineage.

Flow Cytometry Analysis

Cells were analyzed as described (Arrighi et al., 1999), on aFACScalibur (Becton-Dickinson) with slight modifications. FL-1 was usedfor GFP, FL-2 for PE-labeled MAbs, FL-3 for identification of livingcells with the non-permeant DNA dye 7-amino-actinomycin D (Sigma)(Schmid et al., 1994), and FL-4 for biotinylated MAbs indirectly labeledwith streptavidin-APC. Cell suspensions were adjusted to 0.5%paraformaldehyde prior to analysis. Data were analyzed using WINMDIsoftware written by J. Trotter at Scripps Institute (La Jolla, Calif.)and CellQuest software (Becton-Dickinson).

Example 1 Transduction of Human Hematopoietic Progenitors with MLVVectors and HIV-Based Lentivectors

Human cord blood CD34⁺ cells were purified by a single round of positiveselection with magnetic beads, resulting in preparations approximately90% pure. Cells were maintained overnight in serum-free mediumsupplemented with TPO, and were exposed for 24 hrs to MLV- or HIV-basedGFP-expressing vectors pseudotyped with the G protein of vesicularstomatitis virus (VSV G), prepared by transient transfection of 293Tcells as previously described (Naldini et al., 1996a; Zufferey et al.,1997). The lentiviral packaging system used in these experiments was ofa so-called second generation (Zufferey et al., 1997), comprising theHIV-1 gag, pol, tat and rev genes. The multiplicity of infection (MOI)was 10 HeLa-transducing unit (TU) per CD34⁺ cell. After transduction,cells were incubated for another three days in medium containing FCS,FLT3-L, TPO and SCF, and analyzed by flow cytometry for GFP (FIG. 1) andCD34 expression and lysed for PCR analysis. The relative potencies ofthree internal transcription units were compared within the context oflentivectors: the cytomegalovirus immediate early (CMV), thephosphoglycerate kinase (PGK) and the alpha chain of elongation factor1α (EF1α) promoters.

As shown in FIG. 1A, a sharp subpopulation of GFP-positive hematopoieticprogenitors could only be seen when cells were transduced with HIVvectors containing the PGK or the EF1α promoters. Cells transduced withMLV vector or HIV vector containing the CMV promoter displayed only asmall percentage of GFP-positive cells, together with a highheterogeneity in GFP expression. A side-by-side comparison of GFPexpression after transduction of HeLa and CD34⁺ cells revealed that thepromoters examined behaved quite differently in these two cell types.The PGK promoter was weak in HeLa cells (overlap between GFP⁺ and GFP⁻cells) and strong in HPCs. The EF1α promoter was intermediate in HeLacells and very potent in HPCs, with a mean value typically 100 timeshigher in transduced than in control HPCs. The CMV promoter, whether inan MLV or an HIV vector, was equally strong in HeLa cells, contrastingwith its low activity in transduced HPCs. A PCR-based quantificationperformed at the time of the flow cytometry analysis (FIG. 1B) indicatedthat the low expression from the MLV-CMV vector in HPCs was due at leastin part to poor gene transfer in these cells, a consequence of thelimited stimulation of CD34⁺ cells during transduction. In contrast, thelow expression in HPCs exposed to the HIV-CMV vector (FIG. 1A) could notbe explained by a low frequency of transduction, since equivalentamounts of transgene DNA were present in cells transduced with all threeHIV vectors (FIG. 1B). This indicates that the CMV promoter does notgovern efficient expression in hematopoietic progenitors, a finding inaccordance with previous reports (Miyoshi et al., 1999; Case et al.,1999; An et al., 2000).

The inventors also ascertained that GFP expression originated fromintegrated proviruses. For this, a PCR analysis was performed following3 weeks of expansion of the transduced CD34⁺ cells. During this timeperiod, the cell numbers increased by a factor of 900 to 1800, dependingon conditions and experiments. In the particular experiment illustratedhere (FIG. 1B), the percentage of cells expressing GFP from the PGKpromoter decreased by a factor of approximately 2 after expansion anddifferentiation into erythroid or monocytoid cells, which paralleled aslight decrease in GFP DNA copy number. This confirms that the bulk oftransgene DNA remains in the cells after massive expansion, excludingits presence as unintegrated forms. This also correlates the previousdemonstration that long term transgene expression cannot be detectedafter transduction with integrase-deficient HIV-vectors (Naldini et al.,1996b).

Thus, the EF1α promoter induces high transgene expression inlentivector-transduced CD34⁺ cells

Example 2 Transduction of Human CD34⁺ Cells as a Function of IncreasingVector Concentration

The high levels of GFP expression induced by the EF1α-containingHIV-derived vector allowed for a reliable determination of gene transferefficiency, because even low numbers of transduced cells were easilydetected. This vector was thus used to evaluate the influence of the MOIon the transduction efficiency of CD34⁺ cell (FIG. 2). Although thepercentage of GFP⁺ cells initially increased as a direct function of theMOI, the curve flattened starting at a MOI of 5, reaching a maximum of25% GFP⁺ cells (±5% according to experiments) at MOIs 20 and above. Inthe absence of optimal cytokine-induced proliferation, only a fractionof human CD34⁺ cells is thus permissive to lentivector-mediatedtransduction, as previously suggested (Sutton et al., 1999).

Example 3 Transgene Expression in Hematopoietic Lineages afterTransduction of CD34⁺ Cells with HIV Vectors Containing PGK and EF1αPromoters

An important issue for the genetic treatment of a variety oflympho-hematological disorders will be the levels of expression of theputatively therapeutic transgene in the appropriate subset ofdifferentiated cells. In that respect, recent reports using HIV-basedvectors with an internal expression cassette driven by the CMV promoterhave documented the incapacity of this transcriptional element to inducestrong transgene expression in precursors as well as mature blood cells(Miyoshi et al., 1999; Case et al., 1999). To identify more suitablealternatives, hematopoietic progenitors transduced with HIV-basedvectors containing the PGK or the EF1α promoters were differentiated invitro to compare the relative potency of these vectors in varioushematopoietic lineages. For this, transduced CD34⁺ cells were firstexpanded for 7-14 days in FLT3-L/TPO/SCF and then incubated in variousdifferentiating media for an additional 5-to-10-day period, allowing thegeneration of erythroid cells, granulocytes and monocytes.Megakaryocytes were induced in FLT3-L/TPO/SCF exclusively. For dendriticcells (DC), transduced CD34⁺ cells were expanded for 14 to 28 days withFLT3-L/TPO/SCF. Cells were then induced into mature DC by exposure toGM-CSF/IL-4 for 3 days, followed by exposure to GM-CSF/IL-4/TNF for 3more days.

The differentiated cells were then analyzed by flow cytometry todetermine the percentage of lineage-specific marker⁺/GFP⁺ cells, as wellas the relative levels of GFP expression in the differentiatedpopulations (FIG. 3). Transgene expression was high in all lineagesexamined after transduction of precursors with the EF1α-containinglentiviral vector, with a signal-to-noise ratio (mean of fluorescenceintensity of GFP⁺ cells divided by mean of fluorescence intensity ofGFP⁻ cells) comprised between 150 and 200 depending on the cell type.The PGK promoter induced lower and more variable levels of GFPexpression in differentiated cells. Expression of GFP under the controlof the PGK promoter was highest in DCs where it was only 3.5 less potentthan that driven from the EF1α, promoter, and worst in erythroid cells(glycophorin⁺), where it was 11 fold weaker than its EF1α, counterpart.However, even in this case, the signal-to-noise ratio was still highenough to permit a clear discrimination between GFP⁺ and GFP⁻ cells,allowing for the reliable analysis of transgene expression.

Taken together, these data indicate that the transduction of human CD34+cells with HIV-based vectors containing internal promoters derived fromthe EF1α, and to a lesser extent the PGK gene results in high levels oftransgene expression in several hematopoietic lineages.

Example 4 EF1α Promoter-Based Lentiviral Vectors Induce High Levels ofTransgene Expression in Primary T Lymphocytes

The system used in these studies did not allow for the easydifferentiation of CD34⁺ precursors into cells of the lymphocyticlineage. Therefore, to compare the relative value of CMV-, PGK- andEF1α-containing HIV-derived vectors in these cells, mature T-lymphocytespurified from the peripheral blood were directly used as transductiontargets (FIG. 4). The results revealed the same hierarchy as observed inHPCs and in the various other blood cell lineages, with the EF1α,promoter inducing levels of GFP expression significantly higher thanthose yielded by the PGK promoter, whereas cells transduced withCMV-based vectors exhibited levels of GFP expression that wereincompatible with proper detection by flow cytometry. Experiments usinghuman primary B cells show that the CMV promoter is very active in thesecells. This indicates that the poor activity of the CMV promoter is notuniversal in human hematopoietic cells.

Example 5 Influence of SIN Design and WPRE on Transgene Expression

The use of self-inactivating (SIN) 3′LTR U3-deleted HIV vectorsincreases the biosafety of this system and avoids a possibleinterference between the viral LTR and the vector's internal promoter(Zufferey et al., 1998). EF1α- and PGK-containing HIV-derived SINvectors were therefore tested for their ability to induce high levels oftransgene expression in hematopoietic precursors (FIG. 5). The SINdesign was accompanied by a dramatic decrease (6 fold) in GFP expressionwithin the context of the PGK vector, whereas it instead had a slightlyyet reproducibly positive effect when introduced in the EF1α, vector.Inserting the sequence for the woodchuck hepatitis virusposttranscriptional regulatory element (WPRE) (Donello et al., 1998)upstream of the 3′ LTR has been shown to promote transgene expressionfrom HIV-derived vectors in some targets (Zufferey et al., 1999). Itindeed stimulated GFP production from the PGK promoter by a factor of2.15 in hematopoietic precursors. In contrast, it exerted a negativeeffect on expression from the highly active EF1α, promoter, with adecrease by a factor of 1.85. Taken together, these indicate that thestrong EF1α promoter is less sensitive than the PGK promoter to aself-inactivating configuration, making it a better candidate for geneexpression in hematopoietic cells.

All of the compositions and/or methods disclosed and claimed herein canbe made and executed without undue experimentation in light of thepresent disclosure. While the compositions and methods of this inventionhave been described in terms of preferred embodiments, it will beapparent to those of skill in the art that variations may be applied tothe compositions and/or methods and in the steps or in the sequence ofsteps of the method described herein without departing from the concept,spirit and scope of the invention. More specifically, it will beapparent that certain agents which are both chemically andphysiologically related may be substituted for the agents describedherein while the same or similar results would be achieved. All suchsimilar substitutes and modifications apparent to those skilled in theart are deemed to be within the spirit, scope and concept of theinvention as defined by the appended claims.

REFERENCES

The following references, to the extent that they provide exemplaryprocedural or other details supplementary to those set forth herein, arespecifically incorporated herein by reference.

-   U.S. Pat. No. 4,682,195-   U.S. Pat. No. 4,683,202-   U.S. Pat. No. 5,466,468-   U.S. Pat. No. 5,645,897-   U.S. Pat. No. 5,686,279-   U.S. Pat. No. 5,705,629-   U.S. Pat. No. 5,846,225-   U.S. Pat. No. 5,846,233-   U.S. Pat. No. 5,925,565-   U.S. Pat. No. 5,928,906-   U.S. Pat. No. 5,935,819-   U.S. Pat. No. 5,994,136-   U.S. Pat. No. 6,013,516-   U.S. Pat. No. 6,136,597-   EP 266,032-   Akkina, Walton, Chen, Li, Planelles, Chen, “High-efficiency gene    transfer into CD34+ cells with a human immunodeficiency virus type    1-based retroviral vector pseudotyped with vesicular stomatitis    virus envelope glycoprotein G,” J. Virol., 70:2581-2585, 1996.-   Almendro et al., “Cloning of the human platelet endothelial cell    adhesion molecule-1 promoter and its tissue-specific expression.    Structural and functional characterization,” J. Immunol.,    157(12):5411-5421, 1996.-   An, Wersto, Agricola, Metzger, Lu, Amado, Chen, Donahue, “Marking    and gene expression by a lentivirus vector in transplanted human and    nonhuman primate CD34(+) cells,” J. Virol., 74:1286-1295, 2000.-   Angel, Bauman, Stein, Dellus, Rahmsdorf, and Herrlich,    “12-0-tetradecanoyl-phorbol-13-acetate Induction of the Human    Collagenase Gene is Mediated by an Inducible Enhancer Element    Located in the 5′ Flanking Region,” Mol. Cell. Biol., 7:2256, 1987a.-   Angel, Imagawa, Chiu, Stein, Imbra, Rahmsdorf, Jonat, Herrlich, and    Karin, “Phorbol Ester-Inducible Genes Contain a Common cis Element    Recognized by a TPA-Modulated Trans-acting Factor,” Cell, 49:729,    1987b-   Arrighi, Hauser, Chapuis, Zubler, Kindler, “Long-term culture of    human CD34(+) progenitors with FLT3-ligand, thrombopoietin, and stem    cell factor induces extensive amplification of a CD34(−)CD14(−) and    CD34(−)CD14(+) dendritic cell precursor,” Blood, 93:2244-2252, 1999.-   Atchison and Perry, “Tandem Kappa Immunoglobulin Promoters are    Equally Active in the Presence of the Kappa Enhancer: Implications    for Model of Enhancer Function,” Cell, 46:253, 1986.-   Atchison and Perry, “The Role of the Kappa Enhancer and its Binding    Factor NF-kappa B in the Developmental Regulation of Kappa Gene    Transcription,” Cell, 48:121, 1987.-   Banerji et al., “Expression of a Beta-Globin Gene is Enhanced by    Remote SV40 DNA Sequences,” Cell, 27:299, 1981.-   Banerji, Olson, and Schaffner, “A lymphocyte-specific cellular    enhancer is located downstream of the joining region in    immunoglobulin heavy-chain genes,” Cell, 35:729, 1983.-   Berkhout, Silverman, and Jeang, “Tat Trans-activates the Human    Immunodeficiency Virus Through a Nascent RNA Target,” Cell, 59:273,    1989.-   Bhatia, Bonnet, Kapp, Wang, Murdoch, Dick, “Quantitative analysis    reveals expansion of human hematopoietic repopulating cells after    short-term ex vivo culture,” J. Exp. Med., 186:619-624, 1997.-   Blanar, Baldwin, Flavell, and Sharp, “A gamma-interferon-induced    factor that binds the interferon response sequence of the MHC class    I gene, H-2Kb,” EMBO J., 8:1139, 1989.-   Blomer, Naldini, Kafri, Trono, Verma, Gage, “Highly efficient and    sustained gene transfer in adult neurons with a lentivirus    vector,” J. Virol., 71:6641-6649, 1997.-   Bodine and Ley, “An enhancer element lies 3′ to the human a gamma    globin gene,” EMBO J., 6:2997, 1987.-   Boshart, Weber, Jahn, Dorsch-Hasler, Fleckenstein, and Schaffner, “A    very strong enhancer is located upstream of an immediate early gene    of human cytomegalovirus,” Cell, 41:521, 1985.-   Bosze, Thiesen, and Charnay, “A transcriptional enhancer with    specificity for erythroid cells is located in the long terminal    repeat of the friend murine leukemia virus,” EMBO J., 5:1615, 1986.-   Braddock, Chambers, Wilson, Esnouf, Adams, Kingsman, and Kingsman,    “HIV-I Tat activates presynthesized RNA in the nucleus,” Cell,    58:269, 1989.-   Brown, Tiley, Cullen, “Efficient polyadenylation within the human    immunodeficiency virus type 1 long terminal repeat requires flanking    U3-specific sequences,” J. Virol., 65:3340-3343, 1991.-   Bulla and Siddiqui, “The hepatitis B virus enhancer modulates    transcription of the hepatitis B virus surface-antigen gene from an    internal location,” J. Virol., 62:1437, 1986.-   Campbell and Villarreal, “Functional analysis of the individual    enhancer core sequences of polyoma virus: cell-specific uncoupling    of DNA replication from transcription,” Mol. Cell. Biol., 8:1993,    1988.-   Campere and Tilghman, “Postnatal repression of the alpha-fetoprotein    gene is enhancer independent,” Genes and Dev., 3:537, 1989.-   Campo, Spandidos, Lang, Wilkie, “Transcriptional control signals in    the genome of bovine papilloma virus type 1,” Nature, 303:77, 1983.-   Carbonelli et al. “A plasmid vector for isolation of strong    promoters in E. coli,” FEMS Microbiol Lett. 177(1):75-82, 1999.-   Case, Price, Jordan, Yu, Wang, Bauer, Haas, Xu, Stripecke, Naldini,    Kohn, Crooks, “Stable transduction of quiescent CD34(+)CD38(−) human    hematopoietic cells by HIV-1 based lentiviral vectors,” Proc. Natl.    Acad. Sci. USA, 96:2988-2993, 1999.-   Celander and Haseltine, “Glucocorticoid Regulation of Murine    Leukemia Virus Transcription Elements is Specified by Determinants    Within the Viral Enhancer Region,” J. Virology, 61:269, 1987.-   Celander, Hsu, and Haseltine, “Regulatory Elements Within the Murine    Leukemia Virus Enhancer Regions Mediate Glucocorticoid    Responsiveness,” J. Virology, 62:1314, 1988.-   Chandler, Maler, and Yamamoto, “DNA Sequences Bound Specifically by    Glucocorticoid Receptor in vitro Render a Heterologous Promoter    Hormone Responsive in vivo,” Cell, 33:489, 1983.-   Chandler et al., “RNA splicing specificity determined by the    coordinated action of RNA recognition motifs in SR proteins,” Proc    Natl Acad Sci USA. 94(8):3596-3601, 1997.-   Chang, Erwin, and Lee, “Glucose-regulated Protein (GRP94 and GRP78)    Genes Share Common Regulatory Domains and are Coordinately Regulated    by Common Trans-acting Factors,” Mol. Cell. Biol., 9:2153, 1989.-   Chatterjee, Lee, Rentoumis, and Jameson, “Negative Regulation of the    Thyroid-Stimulating Hormone Alpha Gene by Thyroid Hormone: Receptor    Interaction Adjacent to the TATA Box,” Proc Natl. Acad Sci. U.S.A.,    86:9114, 1989.-   Chen and Okayama, “High-efficiency transformation of mammalian cells    by plasmid DNA,” Mol. Cell. Biol. 7:2745-2752, 1987-   Cherrington and Ganem, “Regulation of polyadenylation in human    immunodeficiency virus (HIV): contributions of promoter proximity    and upstream sequences,” Embo. J., 11:1513-1524, 1992.-   Choi, Chen, Kriegler, and Roninson, “An altered pattern of    cross-resistance in multi-drug-resistant human cells results from    spontaneous mutations in the mdr-1 (p-glycoprotein) gene,” Cell,    53:519, 1988.-   Cocea, “Duplication of a region in the multiple cloning site of a    plasmid vector to enhance cloning-mediated addition of restriction    sites to a DNA fragment,” Biotechniques, 23:814-816, 1997.-   Cohen, Walter, and Levinson, “A Repetitive Sequence Element 3′ of    the Human c-Ha-ras1 Gene Has Enhancer Activity,” J. Cell. Physiol.,    5:75, 1987.-   Corbeau, et al., PNAS (U.S.A.,) 93(24):14070-14075, 1996.-   Costa, Lai, Grayson, and Darnell, “The Cell-Specific Enhancer of the    Mouse Transthyretin (Prealbumin) Gene Binds a Common Factor at One    Site and a Liver-Specific Factor(s) at Two Other Sites,” Mol. Cell.    Biol., 8:81, 1988.-   Cripe, Haugen, Turk, Tabatabai, Schmid, Durst, Gissmann, Roman, and    Turek, “Transcriptional Regulation of the Human Papilloma Virus-16    E6-E7 Promoter by a Keratinocyte-Dependent Enhancer, and by Viral E2    Trans-Activator and Repressor Gene Products: Implications for    Cervical Carcinogenesis,” EMBO J., 6:3745, 1987.-   Culotta and Hamer, “Fine Mapping of a Mouse Metallothionein Gene    Metal-Response Element,” Mol. Cell. Biol., 9:1376, 1989.-   Dandolo, Blangy, and Kamen, “Regulation of Polyma Virus    Transcription in Murine Embryonal Carcinoma Cells,” J. Virology,    47:55, 1983.-   Dao, Hannum, Kohn, Nolta, “FLT3 ligand preserves the ability of    human CD34+ progenitors to sustain long-term hematopoiesis in    immune-deficient mice after ex vivo retroviral-mediated    transduction,” Blood, 89:446-456, 1997.-   Dao, Hashino, Kato, Nolta, “Adhesion to fibronectin maintains    regenerative capacity during ex vivo, culture and transduction of    human hematopoietic stem and progenitor cells,” Blood, 92:4612-4621,    1998.-   Deschamps, Meijlink, and Verma, “Identification of a Transcriptional    Enhancer Element Upstream From the Proto-Oncogene Fos,” Science,    230:1174, 1985.-   De Villiers, Schaffner, Tyndall, Lupton, and Kamen, “Polyoma Virus    DNA Replication Requires an Enhancer,” Nature, 312:242, 1984.-   DeZazzo, Kilpatrick, Imperiale, “Involvement of long terminal repeat    U3 sequences overlapping the transcription control region in human    immunodeficiency virus type 1 mRNA 3′ end formation,” Mol. Cell.    Biol., 11:1624-1630, 1991.-   Donello, Loeb, Hope, “Woodchuck hepatitis virus contains a    tripartite posttranscriptional regulatory element,” J. Virol.,    72:5085-5092, 1998.-   Dorrell, Gan, Pereira, Hawley, Dick, “Expansion of human cord blood    CD34(+)CD38(−) cells in ex vivo culture during retroviral    transduction without a corresponding increase in SCID repopulating    cell (SRC) frequency: dissociation of SRC phenotype and function,”    Blood, 95:102-110, 2000.-   Edbrooke, Burt, Cheshire, and Woo, “Identification of cis-acting    sequences responsible for phorbol ester induction of human serum    amyloid a gene expression via a nuclear-factor-kappa β-like    transcription factor,” Mol. Cell. Biol., 9:1908, 1989.-   Edlund, Walker, Barr, and Rutter, “Cell-specific expression of the    rat insulin gene: evidence for role of two distinct 5′ flanking    elements,” Science, 230:912, 1985.-   Fechheimer, Boylan, Parker, Sisken, Patel and Zimmer, “Transfection    of mammalian cells with plasmid DNA by scrape loading and sonication    loading,” Proc Nat'l. Acad. Sci. USA 84:8463-8467, 1987-   Feng and Holland, “HIV-I Tat Trans-Activation Requires the Loop    Sequence Within Tar,” Nature, 334:6178, 1988.-   Firak and Subramanian, “Minimal Transcription Enhancer of Simian    Virus 40 is a 74-Base-Pair Sequence that Has Interacting Domains,”    Mol. Cell. Biol., 6:3667, 1986.-   Foecking and Hofstetter, “Powerful and Versatile Enhancer-Promoter    Unit for Mammalian Expression Vectors,” Gene, 45(1):101-105, 1986.-   Fujita, Shibuya, Hotta, Yamanishi, and Taniguchi, “Interferon-Beta    Gene Regulation: Tandemly Repeated Sequences of a Synthetic 6-bp    Oligomer Function as a Virus-Inducible Enhancer,” Cell, 49:357,    1987.-   Gilles, Morris, Oi, and Tonegawa, “A tissue-specific transcription    enhancer element is located in the major intron of a rearranged    immunoglobulin heavy-chain gene,” Cell, 33:717, 1983.-   Gilmartin, Fleming, Oetjen, “Activation of HIV-1 pre-mRNA 3′    processing in vitro requires both an upstream element and TAR,”    Embo. J., 11:4419-4428, 1992.-   Gloss, Bernard, Seedorf, and Klock, “The Upstream Regulatory Region    of the Human Papilloma Virus-16 Contains an E2 Protein-Independent    Enhancer Which is Specific for Cervical Carcinoma Cells and    Regulated by Glucocorticoid Hormones,” EMBO J., 6:3735, 1987.-   Godbout, Ingram, and Tilghman, “Fine-Structure Mapping of the Three    Mouse Alpha-Fetoprotein Gene Enhancers,” Mol. Cell. Biol., 8:1169,    1988.-   Goodbourn, Burstein, and Maniatis, “The Human Beta-Interferon Gene    Enhancer is Under Negative Control,” Cell, 45:601, 1986.-   Goodbourn and Maniatis, “Overlapping Positive and Negative    Regulatory Domains of the Human β-Interferon Gene,” Proc. Natl.    Acad. Sci. USA, 85:1447, 1988.-   Gopal, “Gene transfer method for transient gene expression, stable    transformation, and cotransformation of suspension cell cultures,”    Mol. Cell. Biol. 5:1188-1190, 1985.-   Gossen and Bujard, Proc. Natl. Acad. Sci., 89:5547-5551, 1992.-   Graham and Van Der Eb, “A new technique for the assay of infectivity    of human adenovirus 5 DNA,” Virology 52:456-467, 1973-   Greene, Bohnlein, and Ballard, “HIV-1, and Normal T-Cell Growth:    Transcriptional Strategies and Surprises,” Immunology Today, 10:272,    1989-   Grosschedl and Baltimore, “Cell-Type Specificity of Immunoglobulin    Gene Expression is Regulated by at Least Three DNA Sequence    Elements,” Cell, 41:885, 1985.-   Haslinger and Karin, “Upstream Promoter Element of the Human    Metallothionein-II Gene Can Act Like an Enhancer Element,” Proc    Natl. Acad. Sci. U.S.A., 82:8572, 1985.-   Hauber and Cullen, “Mutational Analysis of the    Trans-Activation-Responsive Region of the Human Immunodeficiency    Virus Type I Long Terminal Repeat,” J. Virology, 62:673, 1988.-   Hen, Borrelli, Fromental, Sassone-Corsi, and Chambon, “A Mutated    Polyoma Virus Enhancer Which is Active in Undifferentiated Embryonal    Carcinoma Cells is not Repressed by Adenovirus-2 E1A Products,”    Nature, 321:249, 1986.-   Hensel, Meichle, Pfizenmaier, and Kronke, “PMA-Responsive 5′    Flanking Sequences of the Human TNF Gene,” Lymphokine Res., 8:347,    1989.-   Herr and Clarke, “The SV40 Enhancer is Composed of Multiple    Functional Elements That Can Compensate for One Another,” Cell,    45:461, 1986.-   Hirochika, Browker, and Chow, “Enhancers and Trans-Acting E2    Transcriptional Factors of Papilloma Viruses,” J. Virol., 61:2599,    1987.-   Hirsch, Gaugler, Deagostini-Bauzin, Bally-Cuif, and Gordis,    “Identification of Positive and Negative Regulatory Elements    Governing Cell-Type-Specific Expression of the    Neural-Cell-Adhesion-Molecule Gene,” Mol. Cell. Biol., 10:1959,    1990.-   Holbrook, Gulino, and Ruscetti, “cis-Acting Transcriptional    Regulatory Sequences in the Gibbon Ape Leukemia Virus (GALV) Long    Terminal Repeat,” Virology, 157:211, 1987.-   Horlick and Benfield, “The upstream muscle-specific enhancer of the    rat muscle creatine kinase gene is composed of multiple elements,”    Mol. Cell. Biol., 9:2396, 1989.-   Huang, Ostrowski, Berard, and Hagar, “Glucocorticoid regulation of    the ha-musv p21 gene conferred by sequences from mouse mammary tumor    virus,” Cell, 27:245, 1981.-   Hug, Costas, Staeheli, Aebi, and Weissmann, “Organization of the    Murine Mx Gene and Characterization of its Interferon- and    Virus-Inducible Promoter,” Mol. Cell. Biol., 8:3065, 1988.-   Hwang, Lim, and Chae, “Characterization of the S-Phase-Specific    Transcription Regulatory Elements in a DNA-Replication-Independent    Testis-Specific H2B (TH2B) Histone Gene,” Mol. Cell. Biol., 10:585,    1990.-   Imagawa, Chiu, and Karin, “Transcription Factor AP-2 Mediates    Induction by Two Different Signal-Transduction Pathways: Protein    Kinase C and cAMP,” Cell, 51:251, 1987.-   Imbra and Karin, “Phorbol Ester Induces the Transcriptional    Stimulatory Activity of the SV40 Enhancer,” Nature, 323:555, 1986.-   Imler, Lemaire, Wasvlyk, and Waslyk, “Negative Regulation    Contributes to Tissue Specificity of the Immunoglobulin Heavy-Chain    Enhancer,” Mol. Cell. Biol, 7:2558, 1987.-   Imperiale and Nevins, “Adenovirus 5 E2 Transcription Unit: an    E1A-Inducible Promoter with an Essential Element that Functions    Independently of Position or Orientation,” Mol. Cell. Biol., 4:875,    1984.-   Jakobovits, Smith, Jakobovits, and Capon, “A Discrete Element 3′ of    Human Immunodeficiency Virus 1 (HIV-1) and HIV-2 mRNA Initiation    Sites Mediates Transcriptional Activation by an HIV    Trans-Activator,” Mol. Cell. Biol., 8:2555, 1988.-   Jameel and Siddiqui, “The Human Hepatitis B Virus Enhancer Requires    Transacting Cellular Factor(s) for Activity,” Mol. Cell. Biol.,    6:710, 1986.-   Jaynes, Johnson, Buskin, Gartside, and Hauschka, “The Muscle    Creatine Kinase Gene is Regulated by Multiple Upstream Elements,    Including a Muscle-Specific Enhancer,” Mol. Cell. Biol., 8:62, 1988.-   Johnson, Wold, and Hauschka, “Muscle creatine kinase sequence    elements regulating skeletal and cardiac muscle expression in    transgenic mice,” Mol. Cell. Biol., 9:3393, 1989.-   Kadesch and Berg, “Effects of the Position of the Simian Virus 40    Enhancer on Expression of Multiple Transcription Units in a Single    Plasmid,” Mol. Cell. Biol., 6:2593, 1986.-   Karin, Haslinger, Heguy, Dietlin, and Cooke, “Metal-Responsive    Elements Act as Positive Modulators of Human Metallothionein-IIA    Enhancer Activity,” Mol. Cell. Biol., 7:606, 1987.-   Katinka, Yaniv, Vasseur, and Blangy, “Expression of Polyoma Early    Functions in Mouse Embryonal Carcinoma Cells Depends on Sequence    Rearrangements in the Beginning of the Late Region,” Cell, 20:393,    1980.-   Kawamoto, Makino, Niw, Sugiyama, Kimura, Anemura, Nakata, and    Kakunaga, “Identification of the Human Beta-Actin Enhancer and its    Binding Factor,” Mol. Cell. Biol., 8:267, 1988.-   Kiledjian, Su, Kadesch, “Identification and characterization of two    functional domains within the murine heavy-chain enhancer,” Mol.    Cell. Biol., 8:145, 1988.-   Klamut, Gangopadyhay, Worton, and Ray, “Molecular and Functional    Analysis of the Muscle-Specific Promoter Region of the Duchenne    Muscular Dystrophy Gene,” Mol. Cell. Biol., 10:193, 1990.-   Klein et al., “High-velocity microprojectiles for delivering nucleic    acids into living cells,” Nature, 327:70-73, 1987.-   Koch, Benoist, and Mathis, “Anatomy of a new β-cell-specific    enhancer,” Mol. Cell. Biol., 9:303, 1989.-   Kraus et al., “Alternative promoter usage and tissue specific    expression of the mouse somatostatin receptor 2 gene,” FEBS Lett.,    428(3):165-170, 1998.-   Kohn, Nolta, Weinthal, Bahner, Yu, Lilley, Crooks, “Toward gene    therapy for Gaucher disease,” Hum. Gene Ther., 2:101-105, 1991.-   Kriegler and Botchan, “A retrovirus LTR contains a new type of    eukaryotic regulatory element,” In: Eukaryotic Viral Vectors,    Gluzman (ed.), Cold Spring Harbor, Cold Spring Harbor Laboratory, N    Y, 1982.-   Kriegler et al., “Promoter substitution and enhancer augmentation    increases the penetrance of the sv40 a gene to levels comparable to    that of the harvey murine sarcoma virus ras gene in morphologic    transformation,” In: Gene Expression, Alan Liss (Ed.), Hamer and    Rosenberg, New York, 1983.-   Kriegler et al., “Viral Integration and Early Gene Expression Both    Affect the Efficiency of SV40 Transformation of Murine Cells:    Biochemical and Biological Characterization of an SV40 Retrovirus,”    In: Cancer Cells 2/Oncogenes and Viral Genes, Van de Woude et al.    (eds), Cold Spring Harbor, Cold Spring Harbor Laboratory, 1984.-   Kriegler, Perez, Defay, Albert and Liu, “A Novel Form of    TNF/Cachectin Is a Cell-Surface Cytotoxix Transmembrane Protein:    Ramifications for the Complex Physiology of TNF,” Cell, 53:45, 1988.-   Kuhl, De La Fuenta, Chaturvedi, Parinool, Ryals, Meyer, and    Weissman, “Reversible Silencing of Enhancers by Sequences Derived    From the Human IFN-alpha Promoter,” Cell, 50:1057, 1987.-   Kunz, Zimmerman, Heisig, and Heinrich, “Identification of the    Promoter Sequences Involved in the Interleukin-6-Dependent    Expression of the Rat Alpha-2-Macroglobulin Gene,” Nucl. Acids Res.,    17:1121, 1989.-   Lareyre et al., “A 5-kilobase pair promoter fragment of the murine    epididymal retinoic acid-binding protein gene drives the    tissue-specific, cell-specific, and androgen-regulated expression of    a foreign gene in the epididymis of transgenic mice,” J Biol Chem.,    274(12):8282-8290, 1999.-   Larsen, Harney, and Moore, “Repression medaites cell-type-specific    expression of the rat growth hormone gene,” Proc Natl. Acad. Sci.    USA., 83:8283, 1986.-   Laspia, Rice, and Mathews, “HIV-1 Tat protein increases    transcriptional initiation and stabilizes elongation,” Cell, 59:283,    1989.-   Latimer, Berger, and Baumann, “Highly conserved upstream regions of    the alpha.sub.1-antitrypsin gene in two mouse species govern    liver-specific expression by different mechanisms,” Mol. Cell.    Biol., 10:760, 1990.-   Lee, Mulligan, Berg, and Ringold, “Glucocorticoids Regulate    Expression of Dihydrofolate Reductase cDNA in Mouse Mammary Tumor    Virus Chimaeric Plasmids,” Nature, 294:228, 1981.-   Lee et al., “Activation of beta3-adrenoceptors by exogenous dopamine    to lower glucose uptake into rat adipocytes,” J Auton Nerv Syst.    74(2-3):86-90, 1997.-   Levenson et al., “Internal ribosomal entry site-containing    retroviral vectors with green fluorescent protein and drug    resistance markers,” Human Gene Therapy, 9:1233-1236, 1998.-   Levinson, Khoury, VanDeWoude, and Gruss, “Activation of SV40 Genome    by 72-Base-Pair Tandem Repeats of Moloney Sarcoma Virus,” Nature,    295:79, 1982. Lewis and Emerman, “Passage through mitosis is    required for oncoretroviruses but not for the human immunodeficiency    virus,” J. Virol., 68:510-516, 1994.-   Lin, Cross, Halden, Dragos, Toledano, and Leonard, “Delineation of    an enhancer like positive regulatory element in the interleukin-2    receptor.alpha.-chain gene,” Mol. Cell. Biol., 10:850, 1990.-   Luria, Gross, Horowitz, and Givol, “Promoter Enhancer Elements in    the Rearranged Alpha-Chain Gene of the Human T-Cell Receptor,” EMBO    J., 6:3307, 1987.-   Lusky, Berg, Weiher, and Botchan, “Bovine Papilloma Virus Contains    an Activator of Gene Expression at the Distal End of the Early    Transcription Unit,” Mol. Cell. Biol. 3:1108, 1983.-   Lusky and Botchan, “Transient Replication of Bovine Papilloma Virus    Type 1 Plasmids: cis and trans Requirements,” Proc Natl. Acad. Sci.    U.S.A., 83:3609, 1986.-   Majors and Varmus, “A Small Region of the Mouse Mammary Tumor Virus    Long Terminal Repeat Confers Glucocorticoid Hormone Regulation on a    Linked Heterologous Gene,” Proc. Natl. Acad. Sci. U.S.A., 80:5866,    1983.-   Marthas et al. J. Virol., 67:6047-6055, 1993.-   Mazurier, Moreau-Gaudry, Maguer-Satta, Salesse, Pigeonnier-Lagarde,    Ged, Belloc, Lacombe, Mahon, Reiffers, de Verneuil, “Rapid analysis    and efficient selection of human transduced primitive hematopoietic    cells using the humanized S65T green fluorescent protein,” Gene    Ther., 5:556-562, 1998.-   McNeall, Sanchez, Gray, Chesterman, and Sleigh, “Hyperinducible Gene    Expression From a Metallotionein Promoter Containing Additional    Metal-Responsive Elements,” Gene, 76:81, 1989.-   Miksicek, Heber, Schmid, Danesch, Posseckert, Beato, and Schutz,    “Glucocorticoid Responsiveness of the Transcriptional Enhancer of    Moloney Murine Sarcoma Virus,” Cell, 46:203, 1986.-   Miyoshi, Smith, Mosier, Verma, Torbett, “Transduction of human CD34+    cells that mediate long-term engraftment of NOD/SCID mice by HIV    vectors,” Science, 283:682-686, 1999.-   Mizushima and Nagata, “pEF-BOS, a powerful mammalian expression    vector,” Nucleic Acids Res., 18:5322, 1990.-   Mordacq and Linzer, “Co-localization of Elements Required for    Phorbol Ester Stimulation and Glucocorticoid Repression of    Proliferin Gene Expression,” Genes and Dev., 3:760, 1989.-   Moreau, Hen, Wasylyk, Everett, Gaub, and Chambon, “The SV40    base-repair repeat has a striking effect on gene expression both in    sv40 and other chimeric recombinants,” Nucl. Acids Res., 9:6047,    1981.-   Muesing et al., Cell, 48:691, 1987.-   Naldini, Blomer, gallay, Ory, Mulligan, Gage, Verma, Trono, “In vivo    gene delivery and stable transduction of nondividing cells by a    lentiviral vector,” Science, 272:263-267, 1996a.-   Naldini, Blomer, Gage, Trono, Verma, “Efficient transfer,    integration, and sustained long-term expression of the transgene in    adult rat brains injected with a lentiviral vector,” Proc. Natl.    Acad. Sci. USA, 93:11382-11388, 1996b.-   Ng, Gunning, Liu, Leavitt, and Kedes, “Regulation of the Human    Beta-Actin Promoter by Upstream and Intron Domains,” Nuc. Acids    Res., 17:601, 1989.-   Nomoto et al., “Cloning and characterization of the alternative    promoter regions of the human LIMK2 gene responsible for alternative    transcripts with tissue-specific expression,” Gene, 236(2):259-271,    1999.-   Omitz, Hammer, Davison, Brinster, and Palmiter, “Promoter and    enhancer elements from the rat elastase i gene function    independently of each other and of heterologous enhancers,” Mol.    Cell. Biol. 7:3466, 1987.-   Ondek, Sheppard, and Herr, “Discrete Elements Within the SV40    Enhancer Region Display Different Cell-Specific Enhancer    Activities,” EMBO J., 6:1017, 1987.-   Ory et al., Proc. Natl. Acad. Sci., 93:11400-11406, 1996.-   Palmiter, Chen, and Brinster, “Differential regulation of    metallothionein-thymidine kinase fusion genes in transgenic mice and    their offspring,” Cell, 29:701, 1982.-   Pech, Rao, Robbins, and Aaronson, “Functional identification of    regulatory elements within the promoter region of platelet-derived    growth factor 2,” Mol. Cell. Biol., 9:396, 1989.-   Perez-Stable and Constantini, “Roles of fetal γ-globin promoter    elements and the adult β-globin 3′ enhancer in the stage-specific    expression of globin genes,” Mol. Cell. Biol., 10:1116, 1990.-   Piacibello, Sanavio, Severino, Dane, Gammaitoni, Fagioli,    Perissinotto, Cavalloni, Kollet Lapidot, Aglietta, “Engraftment in    nonobese diabetic severe combined immunodeficient mice of human    CD34(+) cord blood cells after ex vivo expansion: evidence for the    amplification and self-renewal of repopulating stem cells,” Blood,    93:3736-3749, 1999.-   Picard and Schaffner, “A Lymphocyte-Specific Enhancer in the Mouse    Immunoglobulin Kappa Gene,” Nature, 307:83, 1984.-   Pinkert, Omitz, Brinster, and Palmiter, “An albumin enhancer located    10 kb upstream functions along with its promoter to direct    efficient, liver-specific expression in transgenic mice,” Genes and    Dev., 1:268, 1987.-   Ponta, Kennedy, Skroch, Hynes, and Groner, “Hormonal Response Region    in the Mouse Mammary Tumor Virus Long Terminal Repeat Can Be    Dissociated From the Proviral Promoter and Has Enhancer Properties,”    Proc. Natl. Acad. Sci. U.S.A., 82:1020, 1985.-   Porton, Zaller, Lieberson, and Eckhardt, “Immunoglobulin heavy-chain    enhancer is required to maintain transfected.gamma.2a gene    expression in a pre-b-cell line,” Mol. Cell. Biol., 10:1076, 1990.-   Potter et al., “Enhancer-dependent expression of human k    immunoglobulin genes introduced into mouse pre-B lymphocytes by    electroporation,” Proc Nat'l Acad. Sci. USA, 81:7161-7165, 1984.-   Queen and Baltimore, “Immunoglobulin Gene Transcription is Activated    by Downstream Sequence Elements,” Cell, 35:741, 1983.-   Quinn, Farina, Gardner, Krutzsch, and Levens, “Multiple components    are required for sequence recognition of the ap1 site in the gibbon    ape leukemia virus enhancer,” Mol. Cell. Biol., 9:4713, 1989.-   Redondo, Hata, Brocklehurst, and Krangel, “A T-Cell-Specific    Transcriptional Enhancer Within the Human T-Cell Receptor .delta.    Locus,” Science, 247:1225, 1990.-   Resendez Jr., Wooden, and Lee, “Identification of highly conserved    regulatory domains and protein-binding sites in the promoters of the    rat and human genes encoding the stress-inducible 78-kilodalton    glucose-regulated protein,” Mol. Cell. Biol., 8:4579, 1988.-   Reisman and Rotter, “Induced Expression From the Moloney Murine    Leukemia Virus Long Terminal Repeat During Differentiation of Human    Myeloid Cells is Mediated Through its Transcriptional Enhancer,”    Mol. Cell. Biol., 9:3571, 1989.-   Remington's Pharmaceutical Sciences, 15^(th) Ed., pages 1035-1038    and 1570-1580.-   Ripe, Lorenzen, Brenner, and Breindl, “Regulatory elements in the 5′    flanking region and the first intron contribute to transcriptional    control of the mouse alpha-1-type collagen gene,” Mol. Cell. Biol.,    9:2224, 1989.-   Rippe, Brenner and Leffert, “DNA-mediated gene transfer into adult    rat hepatocytes in primary culture,” Mol. Cell Biol., 10:689-695,    1990.-   Rittling, Coutinho, Amarm, and Kolbe, “AP-1/jun-binding Sites    Mediate Serum Inducibility of the Human Vimentin Promoter,” Nuc.    Acids Res., 17:1619, 1989.-   Roe, Reynolds, Yu, Brown, “Integration of murine leukemia virus DNA    depends on mitosis,” Embo. J., 12:2099-2108, 1993.-   Rosen, Sodroski, and Haseltine, “The location of cis-acting    regulatory sequences in the human t-cell lymphotropic virus type III    (HTLV-111/LAV) long terminal repeat,” Cell, 41:813, 1988.-   Sakai, Helms, Carlstedt-Duke, Gustafsson, Rottman, and Yamamoto,    “Hormone-Mediated Repression: A Negative Glucocorticoid-Response    Element From the Bovine Prolactin Gene,” Genes and Dev., 2:1144,    1988.-   Sambrook, Fritsch, Maniatis, In: Molecular Cloning: A Laboratory    Manual 2 rev.ed., Cold Spring Harbor, Cold Spring Harbor Laboratory    Press, 1(77):19-17.29, 1989.-   Satake, Furukawa, and Ito, “Biological activities of    oligonucleotides spanning the f9 point mutation within the enhancer    region of polyoma virus DNA,” J. Virology, 62:970, 1988.-   Scharfmann, Axelrod, Verma, “Long-term in vivo expression of    retrovirus-mediated gene transfer in mouse fibroblast implants,”    Proc. Natl. Acad. Sci. USA, 88:4626-4630, 1991.-   Schaffner, Schirm, Muller-Baden, Wever, and Schaffner, “Redundancy    of Information in Enhancers as a Principle of Mammalian    Transcription Control,” J. Mol. Biol., 201:81, 1988.-   Schmid, Uittenbogaart, Keld, Giorgi, “A rapid method for measuring    apoptosis and dual-color immunofluorescence by single laser flow    cytometry,” J. Immunol. Methods, 170:145-157, 1994.-   Searle, Stuart, and Palmiter, “Building a metal-responsive promoter    with synthetic regulatory elements,” Mol. Cell. Biol., 5:1480, 1985.-   Sharp and Marciniak, “HIV Tar: an RNA Enhancer?,” Cell, 59:229,    1989.-   Shaul and Ben-Levy, “Multiple Nuclear Proteins in Liver Cells are    Bound to Hepatitis B Virus Enhancer Element and its Upstream    Sequences,” EMBO J., 6:1913, 1987.-   Sherman, Basta, Moore, Brown, and Ting, “Class II Box Consensus    Sequences in the HLA-DR.alpha. Gene: Transcriptional Function and    Interaction with Nuclear Proteins,” Mol. Cell. Biol., 9:50, 1989.-   Sleigh and Lockett, “SV40 Enhancer Activation During    Retinoic-Acid-Induced Differentiation of F9 Embryonal Carcinoma    Cells,” J. EMBO, 4:3831, 1985.-   Spalholz, Yang, and Howley, “Transactivation of a Bovine Papilloma    Virus Transcriptional Regulatory Element by the E2 Gene Product,”    Cell, 42:183, 1985.-   Spandau and Lee, “Trans-Activation of Viral Enhancers by the    Hepatitis B Virus X Protein,” J. Virology, 62:427, 1988.-   Spandidos and Wilkie, “Host-Specificities of Papilloma Virus,    Moloney Murine Sarcoma Virus and Simian Virus 40 Enhancer    Sequences,” EMBO J., 2:1193, 1983.-   Stephens and Hentschel, “The Bovine Papilloma Virus Genome and its    Uses as a Eukaryotic Vector,” Biochem. J., 248:1, 1987.-   Stuart, Searle, and Palmiter, “Identification of Multiple Metal    Regulatory Elements in Mouse Metallothionein-I Promoter by Assaying    Synthetic Sequences,” Nature, 317:828, 1985.-   Sullivan and Peterlin, “Transcriptional Enhancers in the HLA-DQ    Subregion,” Mol. Cell. Biol., 7:3315, 1987.-   Sutton, Reitsma, Uchida, Brown, “Transduction of human progenitor    hematopoietic stem cells by human immunodeficiency virus type    1-based vectors is cell cycle dependent,” J. Virol., 73:3649-3660,    1999.-   Sutton, Wu, Rigg, Bohnlein, Brown, “Human immunodeficiency virus    type 1 vectors efficiently transduce human hematopoietic stem    cells,” J. Virol., 72:5781-5788, 1998.-   Swartzendruber and Lehman, “Neoplastic Differentiation: Interaction    of Simian Virus 40 and Polyoma Virus with Murine Teratocarcinoma    Cells,” J. Cell. Physiology, 85:179, 1975.-   Takebe, Seiki, Fujisawa, Hoy, Yokota, Arai, Yoshida, and Arai,    “SR.alpha. Promoter: An Efficient and Versatile Mammalian cDNA    Expression System Composed of the Simian Virus 40 Early Promoter and    the R-U5 Segment of Human T-Cell Leukemia Virus Type 1 Long Terminal    Repeat,” Mol. Cell. Biol., 8:466, 1988.-   Taylor and Kingston, “E1A Trans-Activation of Human HSP70 Gene    Promoter Substitution Mutants is Independent of the Composition of    Upstream and TATA Elements,” Mol. Cell. Biol., 10:176, 1990.-   Taylor and Kingston, “Factor Substitution in a Human HSP70 Gene    Promoter: TATA-Dependent and TATA-Independent Interactions,” Mol.    Cell. Biol., 10:165, 1990.-   Taylor, Solomon, Weiner, Paucha, Bradley, and Kingston, “Stimulation    of the Human Heat-Shock Protein 70 Promoter in vitro by Simian Virus    40 Large T Antigen,” J. Biol. Chem., 264:15160, 1989.-   Tavernier, Gheysen, Duerinck, Can Der Heyden, and Fiers, “Deletion    Mapping of the Inducible Promoter of Human IFN-beta Gene,” Nature,    301:634, 1983.-   Thiesen, Bosze, Henry, and Charnay, “A DNA Element Responsible for    the Different Tissue Specificities of Friend and Moloney Retroviral    Enhancers,” J. Virology, 62:614, 1988.-   Tronche, Rollier, Bach, Weiss, and Yaniv, “The Rat Albumin Promoter:    Cooperation with Upstream Elements is Required When Binding of    APF/HNF 1 to the Proximal Element is Partially Impaired by Mutation    or Bacterial Methylation,” Mol. Cell. Biol., 9:4759, 1989.-   Tronche, Rollier, Herbomel, Bach, Cereghini, Weiss, and Yaniv,    “Anatomy of the Rat Albumin Promoter,” Mol. Biol. Med., 7:173, 1990.-   Trudel and Constantini, “A 3′ Enhancer Contributes to the    Stage-Specific Expression of the human Beta-Globin Gene,” Genes and    Dev., 6:954, 1987.-   Tsumaki et al., “Modular arrangement of cartilage- and neural    tissue-specific cis-elements in the mouse alpha2(XI) collagen    promoter,” J Biol Chem. 273(36):22861-22864, 1998.-   Tur-Kaspa, Teicher, Levine, Skoultchi and Shafritz, “Use of    electroporation to introduce biologically active foreign genes into    primary rat hepatocytes,” Mol. Cell Biol., 6:716-718, 1986.-   Tyndall, La Mantia, Thacker, Favaloro, and Kamen, “A Region of the    Polyoma Virus Genome Between the Replication Origin and Late    Protein-Coding Sequences is Required in cis for Both Early Gene    Expression and Viral DNA Replication,” Nuc. Acids. Res., 9:6231,    1981.-   Uchida, Sutton, Friera, He, Reitsma, Chang, Veres, Scollay,    Weissman, “HIV, but not murine leukemia virus, vectors mediate high    efficiency gene transfer into freshly isolated G0/G1 human    hematopoietic stem cells,” Proc. Natl. Acad. Sci. USA,    95:11939-11944, 1998.-   Ueda, Tsuji, Yoshino, Ebihara, Yagasaki, Hisakawa, Mitsui, Manabe,    Tanaka, Kobayashi, Ito, Yasukawa, Nakahata, “Expansion of human    NOD/SCID-repopulating cells by stem cell factor, Flk2/Flt3 ligand,    thrombopoietin, IL-6, and soluble IL-6 receptor,” J. Clin. Invest.,    105:1013-1021, 2000.-   Unutmaz, Kewal, Ramani, Marmon, Littman, “Cytokine signals are    sufficient for HIV-1 infection of resting human T lymphocytes,” J.    Exp. Med., 189:1735-1746, 1999.-   Valsamakis, Schek, Alwine, “Elements upstream of the AAUAAA within    the human immunodeficiency virus polyadenylation signal are required    for efficient polyadenylation in vitro,” Mol. Cell Biol.,    12:3699-3705, 1992.-   Valsamakis, Zeichner, Carswell, Alwine, “The human immunodeficiency    virus type 1 polyadenylylation signal: a 3′ long terminal repeat    element upstream of the AAUAAA necessary for efficient    polyadenylylation,” Proc. Natl. Acad. Sci. USA, 88:2108-2112, 1991.-   Vannice and Levinson, “Properties of the Human Hepatitis B Virus    Enhancer: Position Effects and Cell-Type Nonspecificity,” J.    Virology, 62:1305, 1988.-   Vasseur, Kress, Montreau, and Blangy, “Isolation and    Characterization of Polyoma Virus Mutants Able to Develop in    Multipotential Murine Embryonal Carcinoma Cells,” Proc Natl. Acad.    Sci. U.S.A., 77:1068, 1980.-   Wang and Calame, “SV40 enhancer-binding factors are required at the    establishment but not the maintenance step of enhancer-dependent    transcriptional activation,” Cell, 47:241, 1986.-   Watanabe et al., “Gene transfection of mouse primordial germ cells    in vitro and analysis of their survival and growth control,    Experimental Cell Research, 230:76-83, 1997.-   Weber, De Villiers, and Schaffner, “An SV40 ‘Enhancer Trap’    Incorporates Exogenous Enhancers or Generates Enhancers From its Own    Sequences,” Cell, 36:983, 1984.-   Weinberger, Jat, and Sharp, “Localization of a Repressive Sequence    Contributing to B-cell Specificity in the Immunoglobulin Heavy-Chain    Enhancer,” Mol. Cell. Biol., 8:988, 1984.-   Winoto and Baltimore, “αβ-lineage-specific Expression of the α    T-Cell Receptor Gene by Nearby Silencers,” Cell, 59:649, 1989.-   Wu et al., “Promoter-dependent tissue-specific expressive nature of    imprinting gene, insulin-like growth factor II, in human tissues,”    Biochem Biophys Res Commun. 233(1):221-226, 1997.-   Yang, Burkholder, Roberts, Martinell and McCabe, “In vivo and in    vitro gene transfer to mammalian somatic cells by particle    bombardment,” Proc Nat'l Acad Sci. USA, 87:9568-9572, 1990.-   Yutzey, Kline, and Konieczny, “An Internal Regulatory Element    Controls Troponin I Gene Expression,” Mol. Cell. Biol., 9:1397,    1989.-   Zhao-Emonet et al., “The equine herpes virus 4 thymidine kinase is a    better suicide gene than the human herpes virus 1 thymidine kinase,”    Gene Ther. 6(9):1638-1642, 1999.-   Zufferey, Nagy, Mandel, Naldini, Trono, “Multiply attenuated    lentiviral vector achieves efficient gene delivery in vivo,” Nat.    Biotechnol., 15:871-875, 1997.-   Zufferey, Dull, Mandel, Bukovsky, Quiroz, Naldini, Trono,    “Self-inactivating lentivirus vector for safe and efficient in vivo    gene delivery,” J. Virol., 72:9873-9880, 1998.-   Zufferey, Donello, Trono, Hope, “Woodchuck hepatitis virus    posttranscriptional regulatory element enhances expression of    transgenes delivered by retroviral vectors,” J. Virol.,    73:2886-2892, 1999.

What is claimed is:
 1. A viral particle comprising a recombinantlentiviral vector further comprising: (a) an expression cassettecomprising a transgene positioned under the control of a promoter, otherthan a CMV promoter, that is active to promote detectable transcriptionof the transgene at a signal-to-noise ratio of between about 10 andabout 200 in a T cell; and (b) an LTR region that has reduced promoteractivity relative to wild-type LTR, wherein the LTR region has beenrendered substantially transcriptionally inactive by virtue of deletionsin the U3 region of the 3′ LTR.
 2. The viral particle of claim 1,further comprising an envelope protein isolated from a virus selectedfrom the group consisting of: Moloney murine leukemia virus (MoMuLV orMMLV), Harvey murine sarcoma virus (HaMuSV or HSV), murine mammary tumorvirus (MuMTV or MMTV), gibbon ape leukemia virus (GaLV or GALV), humanimmunodeficiency virus (HIV), Rous sarcoma virus (RSV), and vesicularstomatitis virus (VSV) protein G (VSV G).
 3. The viral particle of claim1, wherein the recombinant lentivirus is further defined as incapable ofreconstituting a wild-type lentivirus through recombination.
 4. Theviral particle of claim 3, wherein the recombinant lentivirus does notexpress a functional lentiviral gene.
 5. The viral particle of claim 1,wherein the promoter is capable of promoting expression of the transgeneat a signal-to-noise ratio of between about 40 and about
 200. 6. Theviral particle of claim 1, wherein the promoter is capable of promotingexpression of the transgene at a signal-to-noise ratio of between about150 and about
 200. 7. The viral particle of claim 1, wherein thepromoter is an EF1α promoter, a PGK promoter, a gp91hox promoter, a WICclass II promoter, a clotting Factor IX promoter, a clotting Factor V111promoter, an insulin promoter, a PDX1 promoter, a CD11 promoter, a CD4promoter, a CD2 promoter or a gp47 promoter.
 8. The viral particle ofclaim 7, wherein the transgene is positioned under the control of theEF1α promoter.
 9. The viral particle of claim 7, wherein the transgeneis positioned under the control of the PGK promoter.
 10. The viralparticle of claim 1, wherein the transgene comprises a nucleic acidencoding erythropoietin, an interleukin, a colony-stimulating factor,integrin αIIbβ, a multidrug resistance gene, gp91hox, gp 47, anantiviral gene, a gene coding for blood coagulation factor VIII, a genecoding for blood coagulation factor IX, a T cell antigen receptor, a Bcell antigen receptor, a single chain antibody (scFv), a fusion protein,TNF, gamma interferon, CTLA4, B7, Melana, or MAGE.
 11. The viralparticle of claim 1, wherein the transgene comprises a nucleic acidencoding Interleukin-2.
 12. The viral particle of claim 1, wherein thetransgene comprises a nucleic acid encoding Interleukin-12.
 13. Theviral particle of claim 1, wherein the transgene comprises a nucleicacid encoding blood coagulation factor VIII.
 14. The viral particle ofclaim 1, wherein the transgene comprises a nucleic acid encoding bloodcoagulation factor IX.
 15. The viral particle of claim 1, wherein thetransgene comprises a nucleic acid encoding MAGE-1.
 16. The viralparticle of claim 1, wherein the transgene comprises a nucleic acidencoding MAGE-3.
 17. The viral particle of claim 1, wherein thetransgene comprises a nucleic acid encoding a T cell antigen receptor.18. The viral particle of claim 1, wherein the transgene comprises anucleic acid encoding a T cell antigen receptor in combination with anscFv.
 19. The viral particle of claim 1, wherein the transgene comprisesa nucleic acid encoding an scFv.
 20. The viral particle of claim 1,wherein the recombinant lentiviral vector further comprises aposttranscriptional regulatory sequence positioned to promote theexpression of the transgene.
 21. The viral particle of claim 20, whereinthe posttranscriptional regulatory sequence is an intron positionedwithin the expression cassette.
 22. The viral particle of claim 21,wherein the intron is positioned in an orientation opposite the vectorgenomic transcript.
 23. The viral particle of claim 20, wherein theposttranscriptional regulatory sequence is a posttranscriptionalregulatory element.
 24. The viral particle of claim 23, wherein theposttranscriptional regulatory element is woodchuck hepatitis virusposttranscriptional regulatory element (WPRE).
 25. The viral particle ofclaim 23, wherein the posttranscriptional regulatory element ishepatitis B virus posttranscriptional regulatory element (HPRE).